This part was designed for the Plug'n'Play with DNA assembly standard (RFC 80), therefore this part contains tails at the 5' and 3' ends as shown on the figure below.
Blue Fluorescent Protein (mTagBFP) without stopcodon
Biobrick part BBa_K592100 of the 2011 iGEM Uppsala team, without stopcodon.
The original stop codon needed to be removed to be able to attach SpyCatcher and His-tag.
Part was developed by iGEM11_Uppsala-Sweden. It was produced by synthesis of part BBa_K1583113
This is a device containing a promoter (BBa_J23106),a RBS (BBa_B0034), a GFP coding sequence (BBa_E0040) and two terminators (BBa_B0010, BBa_B0012) on the pSB1C3, which can be easily transformed into E.coli and produce GFP (mut3b). This is a GFP expression device, which can express GFP (mut3b) without any induction.
To finish the InterLab Study, and want to make it a well characterized device which can be easily used.
Yellow Fluorescent Protein derived from A. victoria GFP. This fluorescent protein is cloned from the pZE12-YFP plasmid used by Elowitz (see reference). The original sequence was made by the The Yeast Resource Center (YRC) based at the University of Washington in Seattle, Washington.
This fluorescent protein was cloned from the pZE12-YFP plasmid used by Elowitz (see reference).
Elowitz, M. B., A. J. Levine, et al. (2002). "Stochastic gene expression in a single cell." Science 297(5584): 1183-6.
N-terminal fragment (Amino acids1-155) of the monomeric[5] cyan fluorescent protein variant ???Cerulean???, designed for fusion/BiFC[1,3]. Reassembly with Split-Cerulean-cCFP (Part Bba_K157005) generates the complete, working CFP ???Cerulean???, whereas combination with the C-terminal fragment (Part Bba_K157007) of the monomeric yellow fluorescent Protein ???Venus???[4] (Part Bba_I757008) results in green fluorescence[2]. - [1] Chang-Deng Hu, Yurii Chinenov, Tom K. Kerppola: ???Visualization of Interactions among bZIP and Rel Family Proteins in Living Cells Using Bimolecular Fluorescence Complementation???, Molecular Cell, Vol. 9, 789???798, April, 2002 - [2] Chang Deng Hu, Tom K. Kerppola: ???Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis???, Nat Biotechnol. 2003 May; 21(5):539-545 (doi:10. 1038/nbt816) -[3] Tom K. Kerppola: ???Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells???, Nat Protoc. 2006;1(3):1278-1286 (doi:10.1038/nprot.2006.201) -[4]Nagai, T. et al. ???A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications??? J. Biol. Chem. 276, 29188-29194, 2001 -[5]Roger Y. Tsien et al. ???Creating new fluorescent probes for cell biology???, Nature Biotechnology Reviews, Vol. 3, 906-918, 2002
Between BioBrick 1.0 sites this part is flanked 5' by NgoMI(=NgoMIV) and 3' AgeI(=PinAI) to facilitate in frame cloning for protein fusions.
Gene synthesis by GeneArt, optimized for expression in homo sapiens.
N-terminal part of the Split-fluorophore CFP, fused to the trans-membrane region of the Erbb1/EGF-receptor.
Derived by cloning parts BBa_K157002, BBa_K157006. For further information see the description of the parts mentioned above.
Readout 1, RFP's transcription controlled by TRP operon
Green Fluorescent Protein with LVA tag (GFPLVA)
GFP with LVA-tag can be degrade by speedy degrager and has a short half-life
UV Mutation Optimized RFP (Improved Expression)
RFP sequence, based on E1010, with the number of possible pyrimidine dimer-forming sites reduced to the maximum possible extent. Altered from BBa_K1673101 to increase effective expression approximately 40%
Sequence produced by the Vanderbilt iGEM 2015 mutation optimization software.
This part accepts POPS to produce EYFP under the strong RBS B0030
This part is a RFC[25]-compatible version of BBa_E0030. The start and stop codons have been removed to make it RFC[25]-compatible and the part is flanked by the RFC[25] prefix- and suffix-sequences.
start and stop-codons have been removed as they are present in the RFC[25] prefix/suffix sequences.
The part is an improved version of BBa_E0030
GFP with a 6X His tag for easier protein purification.
This is the venus YFP, yeast optimized in the Silver Lab BioFusion standard for fusion of protein domains.
gfpmut1 (B. subtilis optimized + Freiburg standard)
This gfpmut3 gene was amplified from pGFPamy B. subtilis vector. It is constructed in the Freiburg standard with RBS. prefix: GAATTCCGCGGCCGCTTCTAGATAAGGAGGAACTACTATGGCCGGC suffix: ACCGGTTAATACTAGTAGCGGCCGCTGCAGT
We designed this part in the Freiburg standard for N- and C-terminal fusions.
EGFP (BBa_1491002) with 6X-His tag for easier protein purification. Original sequence is codon optimized by IDT Codon Optimized Tool for E.coli.
Codon optimized for E.coli and His tag allows for easier protein purification.
David Pohlman, Blythe Ferriere, Aile Able, Erica McFarland
The RBS-3, BBa_B0032, is a medium-strength ribosome bonding site. EYFP, BBa_E0030, is a constituent of GFP, green fluorescence protien. With these two pieces together, this part allows for translation of the EYFP gene.
The two individual parts, BBa_B0032 (RBS-3) and BBa_E0030 (EYFP), came from the iGEM Registry and were subcloned together by our team.
green fluorescent protein derived from A. victoria GFP (SwissProt: P42212) with PEST191 tag
GFP-linker-PEST191 is a part composed of GFP (as a reporter) linked with PEST191 through six aminoacid long protein linker. PEST191 is a sequence that leads towards degradation of protein linked with this sequence. It's estimated half-time is 2h.
GFP-linker-PEST191 is made by PCR overlap extension technique.
KillerRed is a red fluorescent protein that produces reactive oxygen species (ROS) in the presence of yellow-orange light (540-585 nm). KillerRed is engineered from Aequorea victoria GFP (avGFP) to be phototoxic. It has been shown that KillerRed produces superoxide radical anions by reacting with water. Superoxide reacts with the chromophore of KillerRed, causing it to become dark, which ultimately gives rise to a bleaching effect. KillerRed is spectrally similar to mRFP1 with a similar brightness. KillerRed is oligomeric and may form large aggregates. This sequence is codon optimized for mammalian cells and has 14 rare proline codons for E. coli (CCC) and one rare arginine codon (AGA). Codon optimization should be taken into consideration if large amounts of the protein are required. Expression on a high-copy plasmid has produced detectable fluorescent signal, however.
This sequence is not codon optimized for E. coli.
Engineered from avGFP. Provided by the Bruchez lab at Carnegie Mellon
This part is a composite of RiboJ and I13504 which can be used to express GFP if added a promoter.
In order to make other team easily use a GFP part to test the strength of a promoter, we constructed this part contain RiboJ before the BBa_I13504. Any team can use this part to measure the GFP expression if added a promoter.
P2+RBS+GFP+T. P2 can be activated by phosphorylated AgrA.
P2 can be activated by phosphorylated AgrA, leading to the production of GFP and 3OC6HSL. Thus we can detect the presence of S.aureus expediently by observing the expression of GFP.
RFP from Discosoma striata (coral) with DAS-ssrA degradation tag
Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added DAS-ssrA degradation tag (part BBa_M0052). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYADAS and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The DAS tag is reported to have low affinity to ClpX thus its mediated degradation very much depends on the concentration of SspB (ClpX mediator).[1] However, be aware that exact protein degradation rate is influenced by multiple other factors: ClpXP and ClpAP protease concentrations, protein stability, Km of binding to the protease, temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to RFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additonal residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (AAV in this case) are recognised by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
RFP comes from part BBa_E1010, tag sequence was obtained from part BBa_M0052, but same tag was also used in paper by Andersen et al., (1998).[2]
amajLime, yellow-green chromoprotein (incl RBS & J23110)
This chromoprotein from the coral Anemonia majano, amajLime, naturally exhibits strong color when expressed.
Synthesized and codon optimized for E coli by GenScript.
Blue Fluorescent Protein mTagBFP generator (Strong RBS)
Improved version of BBa_K592024 by adding a terminator BBa_B1006 3??? to this part.
RD29A Promoter + Strong Plant Kozak (RBS) + RFP
A split protein is a protein whose sequence has been divided into two (or more) different parts. Often used to study protein-protein interactions, the protein can not perform its function until the parts are put back together. For instance, YFP, the yellow-fluorescent protein, will only express fluorescence when its two parts will be reunited. In normal condition, the production of a protein in response to a stimulus can easily reach several hours due to the many steps required for the protein synthesis. By using split-proteins, we are taking advantage of the absence of fluorescence when the two parts are apart. Indeed, the two parts of our split-YFP, when remaining separated, can be produced without being effective. Therefore, the overall process is far less time-consuming. However, to implement a light control on the fluorescence activation, a genetic construction gathering the VVD photoreceptor and our split-YFP has to be engineered. The new alternative approach for the visualization of protein interactiosn has been developed; the biomolecular fluorescence complementation (BiFC) techniques based on the complementation between fragments of fluorescent proteins; fragments of the yellow fluorescent protein (YFP) brought together by the association of two interaction partners fused to the fragments. They noticed that the spectral characteristics of BiFC of YFP were virtually identical to those of intact YFP.(Chang-Deng Hu, 2003)
Our part consists of an OMP-R binding region that regulates our reporter gene, eYFP. The OMP-R binding region is an omp-c operator, which has three OMP-R binding sites. Our part is regulated by the ENVZ-OMPR two-component regulatory system. ENVZ is a osmolarity sensor on the inner membrane and phosphorylates OMP-R in the presence of high osmolarity. When phosphorylated, OMP-R binds to the omp-c operator and recruits RNAP to transcribe the downstream reporter gene. Fluorescence intensity due to the production of eYFP is a measure of the system's activity. When all three binding sites are occupied by phosphorylated OMP-R proteins, RNAP cannot bind to the DNA and transcribe downstream. This part can be used as an osmolarity indicator. Our team used this part as a glucose sensor because the presence of glucose changes osmolarity. Different concentrations of glucose produce different, quantifiable fluorescence intensities.
Our team had to combine the genes in a specific order. First our team cut out and purified the eYPF gene. We added this gene to a plasmid containing RBS. Next we cut out the RBS+eYFP intermediate and ligated them into the plasmid containing OMP-R. The RBS and OMP-R parts were too small to run out and extract on a gel because they would run off of the end of the gel. Therefore, we had to start the process with eYPF.
This BioBrick was created from parts found in the Registry. These parts were BBa_R0082, BBa_B0032, and BBa_E0030.
C-terminal part of the Split fluorophore YFP fusioned to the transmembrane region of the Erbb1/EGF-receptor with the fluolinker in between.
Derived by cloning of the parts BBa_K157002, BBa_K157009 and BBa_K157007. For further information see the description of the parts mentioned above.
This part has two fused proteins downstream of an arabinose-inducible promoter: CpxR-IFP[1] and CpxR-IFP[2]. This construct was made in all orientations: - IFP[1]-CpxR + IFP[2]-CpxR (BBa_K1286009) - IFP[1]-CpxR + CpxR-IFP[2] (BBa_K1486010) - CpxR-IFP[1] + IFP[2]-CpxR (BBa_K1486011)
All orientations were made, so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used for each of the fused proteins, and an RBS was put before each one of them.
enhanced version of EYFP, yeast-optimized YFP
yeast codon-optimized fast folding variant of YFP
Fuse lambda cI with LGF2 mutated from Asp to Ala at site 78. As one of the components in Bacterial Two-hybrid System, it causes a less interaction with GAL11P compared to the nomalized WT.
This is a composite part including the lambda cI protein and a mutant of LGF2.
Mutated LGF2 at site 78 from Asp to Ala and fused it onto lambda cI.
Tetracycline/aTc inducible promoter with GFP reporter tagged with LVA-ssrA degradation tag followed by terminator. The tagged GFP is actively degraded within cell, thus provides better temporal resolution of green fluorescence and promoter activity.
Part was assembled using BrickClip assembly (BBF RFC104) using other biobrick parts as templates. BrickClip assembly is a special case of more general Paperclip assembly method.[1]
IP-Free BlazeYFP (Yellow Fluorescent Protein)
This part was created by DNA 2.0 as part of their IP-Free series of fluorescent and chromogenic proteins. It is available to use under the BioBrick Public Agreement. Excitation 515nm, Emission 534nm Molecular Weight: 29.2kDA Length: 263aa
This sequence is fully synthetic, (non-Aequorea) and is codon optimized for expression in E. coli.
This synthetic part was created through a gene engineering and synthesis project at DNA 2.0.
Arabinose repressible Tetracycline inversion of yFP with N-terminal Fusion Arganine Tag for targetting into Lumazine Microcompartment. Has a medium RBS and a double terminator.
BioBrick Registry and synthesized Arganine Tag
GFP coding sequence with an N-terminal linker
Designed by Brasil-USP team, BBa_K1819000 is a GFP coding sequence with an N-terminal linker, which is 5???-flanked by NdeI restriction site. It allows to characterize protein expression and secretion, and may serve to other IGEM teams that would like to express a chimera C-terminally attached to GFP, which exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. Such non-usual restriction site, NdeI restriction site, was placed for cloning purposes that lead to expression of recombinant protein with no additional amino acid residue(s) between its coding sequence and the fused N-terminal tag.
As a design consideration we highlight that we chose to add the NdeI restriction site to a GFP coding sequence to avoid any additional amino acid residue between coding sequence and the fused N-terminal tag.
GFP coding sequence source is BBa_E0040 (http://parts.igem.org/Part:BBa_E0040)
This is the second part of the split infrared fluorescent protein (IFP1.4). To be used with IFP[1].
When fused to another coding sequence, insert a flexible linker.
FRET acceptor YPet (optimized to work with CyPet (Cyan FP) as donor
YPet YFP, optimized CyPet (Cyan FP) FRET acceptor
Contains A206K (in this sequence A211K due to N-terminal linker) mutation to reduce dimerization potential as described by Roger Tsien. Contains N- and C-terminal linker sequences (with homology to biobrick parts BBa_E2060 (mCherry), BBa_E2050 (mOrange), and BBa_E2020 (Cerulean CFP) to facilitate color swapping in yeast. Adds N-"MATSG" and "GSGTA"-C. to published amino acid sequence. Inserts a "V" after normal start M. Double TAATAA stop codon. Missing EcoRI, HindIII, NotI, NdeI, XhoI, RsrII, BamHI, NcoI, BglI, SpeI, XbaI, and PstI. Except for 5' and 3' ends no significant sequence identity runs with above biobrick FPs and GFP S65T as found in O'Shea deletion strain collection (originally from plasmid pFA6-GFP(S65T)-His3MX6).
Nguyen et al, (2005) Nature Biotech Feb 6 Epub PMID: 15696158
Calgary's BBa_K339007 repaired (part didn't have the sequence they said they had.
mCherry is a red fluorescent protein used for analysis of localisation of protein.
The IbpB thermometer is an RNA thermometer element found in the ibpAB operon. The operon contains two heat-shock genes, encoding inclusion body binding proteins A and B (IbpA/B), and is the most drastically upregulated operon under heat-shock in Escherichia coli. IbpA is regulated by a ROSE element found in its 5' UTR, while IbpB has its own heat-sensitive cis-regulatory element. The activity of this thermoregulator was confirmed in vitro but was not found in vivo, suggesting more complicated operon regulation exists in bacterial cells. In this part, red fluorescent protein (RFP) is added in the downstream region to measure the activity of RNA thermometer. You check our Experience page for further information about our characterization and experiments. For the inner elements in the part design; check out the Part Design page. RNA Thermometers are the only known single-component regulators of gene expression.(1) RNA Thermometers are thermosensors that regulate gene expression by temperature-induced changes in RNA conformation. Naturally occurring RNA Thermometers exhibit complex secondary structures which are believed to undergo a series of gradual structural changes in response to temperature shifts.(2) RNA Thermometers often regulate genes required during either a heat shock or cold shock response, but have been implicated in other regulatory roles such as in pathogenecity and starvation.(3)
Note that RNA thermometer actually works as an RBS sequence. Therefore, this part does not include an additional ribosome binding site. Also, this part does not contain an terminator sequence, but a simple stop codon. The RFP should be translated continuously.
Escherichia coli, NCBI GeneBank Other elements are obtained from Registry.
A composite part consisting of the signal peptide of Erbb1/EGF-receptor, scFV-antiNIP, GGGSflexlinker (extracellular part), the trans-membrane region of the Erbb1/EGF-receptor and the C-terminal part of the split fluorophor YFP with a fluolinker joined to its N-Terminus (intracellular part).
Derived by cloning of the parts BBa_K157001, BBa_K157004, BBa_K157013, BBa_K157002, BBa_K157009 and BBa_K157005. For further information see the description of the parts mentioned above.
Rock-paper-scissors - Scissors primary plasmid
Composite part, consisting of BBa_K180018 and BBa_K180019, but with BBa_K180018 reading along the reverse strand, and BBa_K180019 reading along the forward strand. Forms the 'scissors' primary plasmid in Team:Southampton's rock-paper-scissors project design.
BBa_K180018 reading along reverse strand, and BBa_K180019 reading along the forward strand to ensure the constitutive promoter BBa_J23119 (present in BBa_K180018) has no effect over mBanana or LuxI expression in BBa_K180019.
See relevant biobrick component parts (BBa_K180018 and BBa_K180019).
Cellulose binding Domain from Cellulomonas Fimi with Reporter GFP
Cellulose binding Domain of C. Fimi Exoglucanase gene with T7, RBS linked with two amino acids (GS) to a GFP (Freiburg-Standard)
CBDcex fused to a his-tagged GFP in a Freiburg-manner
Cloning-vector and [http://partsregistry.org/Part:BBa_K863121 BBa_K863121]
This part is composed of an arabinose-inducible promoter for superfolded GFP attached by a linker to CpxR (on the N terminus of CpxR).
We had to test both attachments, N and C terminus of the CpxR.
The arabinose promoter and the sfGFP were obtained from the registry, while the CpxR was obtained from the E coli genome.
Yeast optimized superfolder GFP C-terminus (215-238)
Derived by cloning of the parts BBa_K157001, BBa_K157004, BBa_K157013, BBa_K157002, BBa_K157006. For further information see the description of the parts mentioned above.
RFP sequence, based on E1010, with the number of oxidation-prone sites reduced to the minimum possible amount.
Jasmin Weber, Michelle Zoeller, Marcus Geissler, Stefan Bohn, Simon Linder, Sebastian Barthel
Lactose/IPTG inducible promoter with RBS, RFP reporter tagged with LAA-ssrA degradation tag and terminator. The tagged RFP is actively degraded within cell, thus provides better temporal resolution of red fluorescence and promoter activity.
Part was assembled using BrickClip assembly (BBF RFC 104) using other biobrick parts as templates. BrickClip assembly is a special case of more general Paperclip assembly method.[1]
The promoter activated by the release of UirR (K1725420) from the transmembrane protein UirS (K1725410) in response to unidirectional UV-A light is upstream of GFP with the B0034 RBS (BBa_I13500). This allows activity in response to UV-A to be measured. For the promoter to be activated UirR and UirS must be present and the cell has to be exposed to UV-A.
The promoter, including the BioBrick prefix and suffix, was ordered as a G-block. It was ligated to I13500 after restriction that was followed by an Azure-A-based gel extraction.
The promoter comes from the genome of the unicellular cyanobacterium Synechocystis sp. PCC6803.
GFP (mut3b) with AAV-ssrA degradation tag
GFP (mut3b) (see part BBa_E0040) with added AAV-ssrA degradation tag (part BBa_I11012). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYAAAV and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The AAV tag is reported to lead to reasonably fast protein degradation, degrading GFP with rate -0.012 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations, protein stability, Km of binding to the protease, temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
GFP comes from part BBa_E0040, tag sequence was obtained from part I11012, but same tag was also used in paper by Andersen et al., (1998).[2]
This part was designed for the Plug'n'Play with DNA assembly standard (RFC 80), therefore this part contains tails at the 5' and 3' ends as shown on the figure below. Please notice that mRFP1-NLS is not the same as BBa_K678030, BBa_K678031, or BBa_K678032.
From p71-mRFP1 of the Center for Microbial Biotechnology plasmid collection at the Technical University of Denmark. The NLS sequence was synthesized.
Green fluorescence protein (mCerulean) WITHOUT stop codon.
Superfolder Blue Fluorescent Protein is a derivative of superfolder Green Fluorescent protein. Compared to eBFP it displays improved stability and strong blue fluorescence. Excitation peak: 380 nm Emission peak: 440 nm. This is N-terminal of sfBFP, that can be used in Bimolecular Fluorescence Complementation system (BiFC).
PartsRegistry BBa_I746909, originally from Aequorea victoria.
'''The dTomato protein is a fluorescent dimer protein that emits orange-red light when it is excited by green-yellow light. The excitation wavelengths and the emission wavelengths don't overlap as much as in other fluorescent proteins, which makes it preferable to use ??? especially in self‑made fluorometry tests.'''
dTomato was derived from a monomeric mutant of the anthozoan coral RFP, DsRed.
mRFP (DsRed) derived, altered to be a biobrick by removing a PstI site and adding in the ends.
Made a point mutation to eliminate a PstI site in the middle and then added BioBrick ends with the LVA tag using PCR. Sequenced using primers that bind to the pSB1A2 plasmid on either side of the brick.
mCherry from Roger Y. Tsien's lab, altered to be BioBrick compatible and with an LVA tag added.
This part accepts POPS to produce ECFP under the medium RBS B0032
This is the mCherry fluorescent protein, yeast-codon optimized and biobricked in the silver lab BioFusion standard for use in fusion proteins.
mTagBFP with 6x His Tag for protein purification
Lactose/IPTG inducible promoter wuth RFP reporter tagged with AAV-ssrA degradation tag followed by terminator. The tagged RFP is actively degraded within cell, thus provides better temporal resolution of red fluorescence and promoter activity. Part was assembled using BrickClip assembly (BBF RFC 104) using other biobrick parts as templates. BrickClip assembly is a special case of more general Paperclip assembly method.[1] Reference [1] Trubitsyna, M., Michlewski, G., Cai, Y., Elfick, A. & French, C. E. PaperClip: rapid multi-part DNA assembly from existing libraries. Nucleic Acids Res. 1???8 (2014). doi:10.1093/nar/gku829
enhanced green fluorescent protein for use in mammalian cells
Compatible with the Plug'n'Play assembly standard
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with intermediate 6 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
enhanced yellow fluorescent protein derived from A. victoria GFP
Fusion Protein of S-Layer SbpA and mCerulean
Fusion Protein of S-Layer SgsE and mCerulean
Fusion Protein of BBa_K525403 and BBa_J18930
Monomeric red fluorescent protein (mRFP1) is a derivative of the red fluorescent protein cloned from Discosoma coral (DsRed) (1). The successful use of mRFP1 in Aspergillus nidulans has previously been described (2). This part does not contain a stop codon. {| border="1" !colspan="8"|Spectroscopic data (1) |- |Excitation maximum |Emission maximum |Extinction coefficient per chain |Fluorescence quantum yield |pKa |t0.5 for maturation at 37??C |colspan="2"|Rate of photobleach relative to EGFP |- |584 nm |607 nm |44,000 M-1cm-1 |0.25 |4.5 |<1h |fast 7.2 |slow 0.16 |}
This part was designed for the Plug'n'Play with DNA assembly standard (RFC 80), therefore this part contains tails at the 5' and 3' ends as shown on the figure below. Please notice that mRFP1-module is not the same as BBa_K678030 or BBa_K678032.
From p71-mRFP1 of the Center for Microbial Biotechnology plasmid collection at the Technical University of Denmark
enhanced version of EYFP, yeast-optimized
Venus YFP, yeast codon optimized. EX515/EM528
Contains N- and C-terminal linker sequences (with homology to biobrick parts BBa_E2060 (mCherry), BBa_E2050 (mOrange), and BBa_E2020 (Cerulean CFP) to facilitate color swapping in yeast. Adds N-"MATSG" and "GSGTA"-C. to published amino acid sequence. Inserts a "V" after normal start M. Double TAATAA stop codon. Missing EcoRI, HindIII, NotI, NdeI, XhoI, RsrII, BamHI, NcoI, BglI, SpeI, XbaI, and PstI. Except for 5' and 3' ends no significant sequence identity runs with Cerulean CFP (BBa_E2020) or to GFP S65T as found in O'Shea deletion strain collection (originally from plasmid pFA6-GFP(S65T)-His3MX6).
TRP promoter - RBS - TetR - Term - TetR promoter - RBS - RFP - Term
TRP inhibits TetR's transcription while TetR inhibits RFP's transcription.
BBa_I13507 for RFP and BBa_I6007 for TetR
C-terminal part of the Split-fluorophore CFP, fused to the trans-membrane region of the Erbb1/EGF-receptor with the fluo-linker in between.
Derived by cloning of the parts BBa_K157002, BBa_K157009 and BBa_K157005. For further information see the description of the parts mentioned above.
Design considerations This part was designed for the Plug'n'Play with DNA assembly standard (RFC 80), therefore this part contains linkers at the 5' and 3' ends as shown on the figure below.
ECFP with Clontech RBS, no degradation tail
dsRED (BBa_E1010) compatible with Genabler assembly
dsRED (BBa_E1010) part with inward facing EarI recognition sites at the ends
To be compatible with Genabler assembly, the part has inward-facing EarI sites at the ends. When assembled by BioBrick assembly, five N-terminal residues MetAlaSerSerSer- and one C-terminal Ala residue will be added to the original sequence of BBa_E1010.
This part is the twin of the non-compatible biobrick BBa_K1486008 rendered compatible by mutating the two illegal restriction sites. However, all experiments were run on the aforementioned biobrick.
Site-directed mutagenesis was used to remove the restriction sites in the sequence.
http://www.addgene.org/browse/article/8177/ which was then mutated by us.
N-terminal fragment (Amino acids1-156) of the monomeric[5] yellow fluorescent protein variant ???Venus??? [4], designed for fusion/BiFC[1,3]. Reassembly with Part Bba_K157007 (Split-Venus-cYFP) generates the complete, working YFP ???Venus???, whereas combination with the C-terminal fragment (Part Bba_K157005) of the monomeric cyan fluorescent Protein ???CeruleanR206K??? (Part Bba_I757009) results in green fluorescence[2].See "Part Design" for references.
Lux pL controlled LuxR + lux pR autoinducing LuxI (lva tag) + lux pR controlled GFP.
Yichong wang, Rui chen, Xihao liao and Junjie zhang
BBa_K1072000 three protein generators: lux pl-RBS-C0062-B0015 produces LuxR. LuxR forms a complex with the quorum sensing molecule AHL and this complex subsequently increases the transcriptional rate of the lux pR promoter while also decreasing the transcriptional rate of the lux pL promoter. Both of the other generators are under control of this lux pR promoter. The second generator ???luxpR-RBS-J04031-B0015???has a low basal expression of the GFP, which can - of course - be monitored. The third generator produces luxI, which produces the quorum sensing molecule AHL. In combination with LuxR, AHL thus increases the production of AHL and GFP. This part is an improvement of BBa_K546000 and K546005 made by 2011 iGEM11_Wageningen_UR team. A LuxpR promoter, RBS, GFP and double terminator has been added.
constructing device compositing oscillator, coupling, reporter and productor.This part just is the oscillator and reporter.
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 9 AA linker as spacer (BBa_K1680001). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
GFPmut3* has a couple of mutations from wtGFP - S2R and S65G and S72A. Emerald and EGFP have S65T and multiple other mutations. The S2R mutation should have no effect.
1. Elowitz MB and Leibler S. A synthetic oscillatory network of transcriptional regulators. Nature 2000 Jan 20; 403(6767) 335-8. doi:10.1038/35002125 pmid:10659856. PubMed HubMed [Cormack]
dnMyD88 is a dominant negative form of protein MyD88 (an important protein in signaling cascade through TLR4 receptor). dnMyD88 is linked to GFP (acts as a reporter) through six aminoacid long protein linker.
dnMyD88-linker-GFP was made by using PCR overlap extension technique. This part is cloned in BioBrick vector pSB1AK3. Original sequence has PstI restriction site so we have to introduce a mutation without affect on amino acid sequence.
With Plux gene,it can detect the concentration of AHL-LuxR protein complex and initiate the following gene Sequence and Features
Celia Chenebault, Camille Soucies, Benjamin Piot
mTagBFP (BBa_K1491001) with a 6X-His tag for easier protein purification. The sequence was codon optimized by IDT Sequencing for E.coli.
Sequence was codon optimized by IDT sequencing for E.coli.
sfGFP in Freiburg format (RFC 25) to allow for easy use in fusion proteins.
Built by Josh as an intermediate in screening plasmid construction
This is the coding sequence of a yeast optimized GFP with BD ends for AarI cloning.
As an AD part, there is no ATG, and the stop codon is included. For more information on AarI cloning, please refer to the UCSF 2008 igem page.
C-terminal fragment (Amino acids156-236) of the monomeric[5] cyan fluorescent protein variant ???Cerulean???, designed for fusion/BiFC[1,3]. Reassembly with Split-Cerulean-nCFP (Part Bba_K157006) generates the complete, working CFP ???Cerulean???, whereas combination with the N-terminal fragment (Part Bba_K157008) of the monomeric yellow fluorescent Protein ???Venus???[4] (Part Bba_I757008) results in green fluorescence[2].References see "Part Design".
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (Freiburg-Standard)
Untagged version of gfp from Repressilator reporter. See the [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0040 untagged version] for source information.
Added Freiburg-restriction-sites for in frame assembly.
[http://partsregistry.org/Part:BBa_I13522 BBa_I13522]
This is the coding sequence for mCherry with AB ends for Aar1 cloning.
Since this is an BD part, there is no ATG but there is a stop codon. For more information on AarI cloning, please refer to the UCSF 2008 igem page.
Fusion Protein of S-Layer SgsE and mCherry RFP
Fusion Protein of BBa_K525303 and BBa_J18932
**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral) with barcode
Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena Büscher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Schäfer, Carolin Schmelas, Silvan Schmitz, Max Waldha
This part is essentially identical to BBa_E1010 , but has a point mutation in the barcode. Barcodes are discontinued, but composite parts using this part will have the mutated barcode.
This is coding sequence of GFP (yeast optimized codons).
This is the first part of the split infrared fluorescent protein (IFP1.4). To be used with IFP[2].
CFP with targeting sequence and LVA tag. The targeting sequence targets the protein to the encapsulin nanocompartment [1] with the aim of increasing the half-life of the protein. LVA should help limit the amount of background activity. [1] M. Sutter, et al., "Structural basis of enzyme encapsulation into a bacterial nanocompartment," Nat. Struct. & Mol. Biol., vol. 15, pp. 939-47., 2008.
We decided to position the LVA at the C-terminus of the construct since that is its natural position. However, the tgt extension sequence is also typically found at the C-terminus, so we're hoping that the shorter LVA sequence won't interfere too much with the binding with encapsulin. If necessary, it'll be possible to remove the LVA tag via PCR.
This BioBrick is for testing the virA/G signaling system. The read-out is the mRFP E1010 . This system contains an unmutated ''virA'', so it is possible to measure, how the natural virA/G system works and reacts.
this is only for measuring the natural virA/G system
''A. tumefaciens'' C58 TI-plasmid, partsregistry, Mr. Gene
Part consists of the signal peptide of Erbb1/EGF-receptor, scFV-antiNIP, GGGSflexlinker (extracellular part), the trans-membrane region of the Erbb1/EGF-receptor and the C-terminal part of the split fluorophor YFP with a fluolinker joined to its N-Terminus (intracellular part).
Derived by cloning of the parts BBa_K157001, BBa_K157003, BBa_K157013, BBa_K157002, BBa_K157009 and BBa_K157005. For further information see the description of the parts mentioned above.
CherryNLS - synthetic construct monomeric red fluorescent protein with nuclear localization sequence
This protein is used to show that T7 polymerase is actually going into the nucleolus, where it can transcribe genes under T7 promotor.
In the original cherry protein there was a PstI restriction site in the middle of the sequence. That is why mutagenesis of this restiction site was necessary.
From one clone that Gabi gave me - will be further described in the future.
Constitutive promoter (J23106) and GFP (S05308)
Constitutive promotor from Anderson library (BBa_J23106) assembled to green fluorescent protein attached to linker (BBa_S05308). This part was used to characterize BBa_K1819000 and compare its efficiency to green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (BBa_E0040).
This part results from the assembly of BBa_J23106 and BBa_S05308
Strong reporter device optimized for Pichia pastoris, activated by methanol
This device is composed by three parts. The first part is a modified promoter pAOX, it was found in a library where it showed more than 60% stronger activation when compared with the wild-type, as reported by Hartner FS, Ruth C, Langenegger D, Johnson SN, Hyka P, Lin-Cereghino GP, Lin-Cereghino J, Kovar K, Cregg JM, Glieder A: Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic Acids Res 2008, 36:e76. The second part is the kozak sequence for Pichia pastoris, and the third is a Red fluorescent protein condon optimized for expression in Pichia pastoris. This device was created to express a reporter gene in the presence of ethanol, and is inactivated by ethanol, when combined with the modified Mxr1 (BBa_K1078000), it is not inactivated by ethanol. This is useful for our biosensor design, which aims to detect levels of methanol above 2% in common alcoholic drinks (normal content of 10 to 60% ethanol). This will allow government to make high-throughput screening of ethanol drinks tainted will methanol. The modified Mxr1 is able to activate the pAOX promoter, in ethanol, glycerol and methanol. This is useful for our biosensor design, which aims to detect levels of methanol above 2% in common alcoholic drinks (normal content of 10 to 60% ethanol). This will allow government to make high-throughput screening of ethanol drinks tainted will methanol.
After synthesis, in order to build our sensor the part was introduced in pPIC9K plasmid to allow its genomic recombination into the Pichia pastoris genome, substituting the original Mxr1 found in the yeast. This part does not have a single biobrick version with the Mx1p because both must be inserted in different genome locations at the Pichia pastoris
he part was obtained by synthesis, the modified Mxr1p sequence was found in Hartner FS, Ruth C, Langenegger D, Johnson SN, Hyka P, Lin-Cereghino GP, Lin-Cereghino J, Kovar K, Cregg JM, Glieder A: Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic Acids Res 2008, 36:e76
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 15 AA linker as spacer (BBa_K1680003). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
amilGFP, yellow chromoprotein (incl RBS)
This chromoprotein from the coral Acropora millepora, amilGFP, naturally exhibits strong yellow color when expressed. The color is readily visible to naked eye both in LB-culture and on agar plates. Color development can be seen in less than 24 hours of incubation.
Two illegal internal restriction sites (EcoRI and PstI) were removed.
Acropora millepora. The DNA was provided by Jeffrey Miller at UCLA, and was made BioBrick-compatible by removing two illegal internal restriction sites (EcoRI and PstI).
Game of Life - Primary plasmid (part 2) [lux pR, GFP and LacI generator]
Part of the iGEM2009 Team:Southampton Game of Life primary plasmid.
Regulated expression of LacI (C0012) and GFP (E0040) by PLUX promoter (R0062), controlled by presence/absence of AHL-LuxR.
See relevant basic biobrick parts (R0062, C0012, B0034, K180001, B0015)
Yeast Optimized sfGFP-N + ADH1 terminator + Kanamycin resistance
Superfolder Cyan Fluorescent Protein is a derivative of superfolder Green Fluorescent protein. Compared to CFP it displays improved stability and strong cyan fluorescence. Excitation peak: 425 nm Emission peak: 475 nm. This is N-terminal of sfCFP, that can be used in Bimolecular Fluorescence Complementation system (BiFC).
RFP (BBa_1491008) with 6X His Tag for easier protein purification. Original sequence was codon optimized for E.coli by IDT Codon optimization tool.
His tag for easier protein purification and codon optimized for E.coli.
A composite part consisting of the signal peptide of Erbb1/EGF-receptor, which ensures the transport to the cell membrane scFV-antiNIP, GGGSflexlinker (extracellular parts), the trans-membrane region of the Erbb1/EGF-receptor and the N-terminal part of the split fluorophor YFP(intracellular part).
Derived by cloning of the parts BBa_K157001, BBa_K157004, BBa_K157013, BBa_K157002, BBa_K157008. For further information see the description of the parts mentioned above.
Tetracycline/aTc inducible promoter with GFP reporter tagged with NYADAS-ssrA degradation tag followed by terminator. The tagged GFP is actively degraded within cell, thus provides better temporal resolution of green fluorescence and promoter activity.
engineered cyan fluorescent protein derived from A. victoria GFP
The dTomato protein is a fluorescent dimer protein that emits orange-red light when it is excited by green-yellow light. It is preferable to use ??? especially in self-made fluorometry tests ??? because the excitation wavelengths and the emission wavelengths do not overlap as much as in other fluorescent proteins. The dTomato excitation peak is at 554 nm and 50% of it is at 510 nm. The emission peak is at 581 nm and the 50% emission peak is at 629 nm. Characterization has been made with the Anderson promoters BBa_J23101 (this construct) and BBa_J23110 (construct BBa_K1688011)
The part is basically a sequence encoding red florescent protein. With specially designed primers, it can be fused with its previous signal protein, yebF.
It is RFC23 compatible, with specially designed prefix.
Obtaining this sequence, we designed primers to take the sequence from IGEM distributed part, BBa_E1010.
Lactose/IPTG inducible promoter with RFP reporter tagged with DAS-ssrA degradation tag followed by terminator. The tagged RFP is degraded within cell, thus provides better temporal resolution of red fluorescence and promoter activity. Presence of SspB is crucial for effective protein degradation.
**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral)
monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm
TAATAA double stop codon added (DE). Four silent mutations made to remove three EcoRI sites and one PstI site: A28G, A76G, A349G, G337A.
Campbell et al., PNAS v99 p7877 URL
This part accepts POPS to produce EYFP under the weak RBS B0031
Digestion/ligation of Pr-RBS with GFP reporter (BBa_I13401)
GFP is presented outside E. Coli as a positive control for protein presentation
His tag is inserted between OmpA and GFP for easier detection in Western Blotting.
OmpA is cloned from BL21(DE3) E. Coli strain. GFP is from Clontech plasmid pGFP-N1
Vector expressing eGFP from the CMV promoter with the BGH polyA tail and with a hygromycin resistance cassette
Compatible with the Plug'n'Play with DNA assembly system.
This part is the Amino Half of GFP with termination codon
Because of the need of our project, we added a termination codon when designing the PCR.
This part is gained from part BBa_I715019. we added the termination codon when designing the PCR.
Fusion Protein of BBa_K525303 and BBa_J18930
pCopA TAT Csp1 sfGFP with divergent CueR
pCopA TAT Csp1 sfGFP with plasmid constitutively expressed CueR
Derived by cloning of the parts BBa_K157001, BBa_K157003, BBa_K157013, BBa_K157002, BBa_K157009 and BBa_K157007. For further information see the description of the parts mentioned above.
Highly engineered monomeric mutant of RFP. Improved version of part BBa_E1010 to remove illegal AgeI sites and add RFC25 prefix and suffix.
Codon optimised for E. coli. RFC 25 compatible.
mRFP highly engineered variant originally from Discosoma striata.
Pu promoter and RBS from GLuc coding device with Pu (BBa_K2023003) and GFP reporter for RHS of library test constructs (BBa_I13401)
CFP protein generator without transcription terminator
Protein generator of cyan fluorescent protein without transcription terminator. It may be negative regulated by Mnt repressor (BBa_C0072).
Codon optimization for better expression in E. coli. Illegal sites were removed.
EYFP with Clontech RBS, no degradation tail
Blue Fluorescent Protein mTagBFP generator (Medium RBS)
Improved version of BBa_K592023 by adding a terminator BBa_B1006 3???to the part.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
Reo Yoshimura,Toshihiro Matsunami,Takahiro Nakagawa,Jyunpei Yokoigawa
This plasmid vector was designed to fuse GFP to C-terminal regions of any proteins.(We use BBa_E0240.) In our projects, we use this plasmid to fuse GFP to dMLF, DIAP2, API2-MALT1 and others to monitor expression of these proteins in E. coli and Drosophila.
Primer sequence F:TTGAATTCTTTTCTAGATTTCGTAAAGGAGAAGAA R:AAACTGCAGAAAACTAGTTTTTTATTATTTGTATAG
820base sequence AAACCCATAATATCATGACATACCCTATAAAAATAGGCGTATCACGAGGCAGAATTCAGATAAAAAAAATCCCTAGCTTTCGCTAAGGATGATTCTGGAATTCTTTCTAGATTCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATTTTAATCT
Variant of GFP that folds robustly even when fused to poorly folded proteins. Waldo and other research groups has reported the engineering of a superfolder GFP (sfGFP) that showed increased resistance to denaturation, improved folding kinetics, and increased resistance to aggregation during refolding (Pedelacq et al., 2006; Andrews et al., 2007; Fisher and DeLisa, 2008). sfGFP has proven to be very useful as a scaffold for improved protein detection and tagging both in vivo and in vitro using self-assembled sfGFP fragments (Cabantous et al., 2005b; Cabantous and Waldo, 2006). Furthermore, sfGFP fusions are more soluble than conventional GFP fusions (Wu et al., 2009).
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 12 AA linker as spacer (BBa_K1680002). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
Based on the part E0040, but with an LVA degradation tail attached.
LVA degradation tail and prefix/suffix addition by sequential PCR amplifications with specially designed primers.
Based upon GFP from E0040, with LVA degradation tail attached.
This part produces GFP in the presence of T3 RNA polymerase. It contains a T3 promoter, a strong RBS, the coding region for GFP, and a double terminator. The GFP is a mutant known as GFPmut3b, and the original citation is as follows: Cormack, B.P., Valdivia, R.H., and S. Falkow. FACS-optimized mutants of green fluorescent protein (GFP). Gene 173: 33-38 (1996). (http://www.sciencedirect.com/science/article/pii/0378111995006850) It has a maximum excitation at 501 nm and maximum emission at 511 nm. For more information, see part BBa_E0040.
The part containing GFP was cut with EcoRI and XbaI, and ligated with the annealed oligos to form the completed part. Note that due to the choice of cloning technique, a Biobrick scar site remains between the promoter and RBS.
The RBS-GFP-terminator was obtained from as part BBa_E0840 from the 2015 Distribution (Plate 2, Well 24D). The T3 promoter was obtained as two short oligonucleotides annealed to form sticky ends. The part containing GFP was then cut with EcoRI and XbaI, and ligated with the annealed oligos to form the completed part.
This part codes for the bright blue fluorescent protein mTagBFP. mTagBFP is a monomeric protein with a narrow fluorescence emission spectrum with a maximum at 456 nm. It has a tyrosine-based chromophore, giving it substantially higher brightness, faster chromophore maturation and higher pH stability than blue fluorescent proteins with a histidine in the chromophore. Subach et. al. started with a red fluorescent protein (TagRed) and converted it to a bright blue monomeric protein. See the article listed in source. The sequence has been codon optimized for expression in E coli by DNA 2.0.
The sequence has been codon optimized for expression in E coli by DNA 2.0.
Subach, O. M., I. S. Gundorov, et al. (2008). "Conversion of red fluorescent protein into a bright blue probe." Chem Biol 15(10): 1116-24.
This is designed as the positive control for protein presentation on the outer membrane.
His tag is inserted between OmpA and mCherry to facilitate detection after expression.
OmpA is cloned from genomic DNA of BL21(DE3) E. Coli strain. mCherry is from the Clontech plasmid pmCherry-C1.
Cellulose binding Domain of C. cellulovorans cellulose binding protein with Reporter GFP
Cellulose binding Domain from Clostridium cellulovorans [http://www.ncbi.nlm.nih.gov/nuccore/M73817 cellulose binding protein gene] with T7, RBS linked with two amino acids (GS) to a GFP with His-tag.
CBDclos fused to a his-tagged GFP in a Freiburg-manner
This part accepts POPS to produce ECFP under the strong RBS B0030
YFP (BBa_K1491004) with 6X-His tag for easier protein purification. Sequence was codon optimized for E.coli via IDT codon optimizing tool.
Codon optimized for E.coli and 6X-His tag for easier protein purification.
fwYellow, yellow chromoprotein (incl RBS)
This chromoprotein naturally exhibits strong color when expressed.
Cloned from FezziwigYFP vector, DNA 2.0.
Partially optimized RFP sequence, based on E1010. Has a 4% reduction in the number of possible pyrimidine dimer-forming sites.
This part is a reporter sfGFP(Sp) originally optimized for Streptococcus pneumoniae.
sfGFP originally optimized for streptococcus pneumonia because it could be shown that the signal in Bacillus is even brighter than the one from sfGFP(Bs).
Fluorescent ratiometric construct for standardizing promoter output
Biosensors may give unreliable outputs. This is due to differences in the number and state of the cells from test to test. By including an internal control signal, to which another inducible signal may be normalised, the reliability and reproducibility of a sensor may be significantly improved. The construct that uses an inducible eCFP (E0020) and a constitutively expressed eYFP (E0030) under the control of the constitutive promoter Pveg (K143012). The construct is optimized in both E. coli and B. subtilis, through the use of B. subtilis ribosome binding sites GsiB (K143020) and SpoVG (K143021).
This biobrick is best used with a shuttle vector that can function in both E. coli and B. subtilis, which should also have preferably different antibiotic markers in E. coli and B. subtilis.
This biobrick is composed entirely of existing biobricks: GsiB (K143020), eCFP (E0020), terminator (B0015), Pveg + SpoVG (K143053), eYFP (E0030) and a second terminator (B0010). It was constructed first on the E. coli- B. subitilis shuttle vector pJS130, then moved onto pSB1C3 for submission.
a fast degrading GFP ligated to the rear of a sulA promoter in 408 form (only recognizable by our LexA-VVD fusing protein, not by E.coli endogenous LexA). The sulA promoter promotes a gene which express SulA protein, a differentiation inhibitor, and was a member of the SOS regulon family. When co-transformed with our luminesensor plasmid into E.coli cell illuminated by blue light, the light will triger the dimerizaiton of the LexA408-VVD fusion protein and the dimerized LexA408 domain will bind the SOS box in 408 form in the SulA408 promoter to inhibit the transcription of the downstream gene so that no GFP will be expressed; if the environment is dark, the luminesensor will not dimerize and no supression of the promoter will occour, and GFP will be expressed.
GFP ligated to the rear of a sulA promoter in 408 form (only recognizable by our LexA-VVD fusing protein, not by E.coli endogenous LexA).
GFP with ssrA tag was from fromer Peking iGEM team members.
RFP sequence, based on E1010, with the number of possible pyrimidine dimer-forming sites reduced to the maximum possible extent.
This part is similar to [http://partsregistry.org/Part:BBa_J61002 J61002] but the backbone is based on pSB1C3 and carries a chloramphenicol resistance.
* This part is a compination of E1010, B0034 and pSB1C3 from the registry.
This is a construct for testing promoter strength by measuring GFP fluorescence.
This part is a generator for RFP under the prosseA promoter
The prosseA promoter is self-designed The rbs, RFP and the terminator are from the registry
We used this construct to test pBAD strong promoter strength by measuring RFP fluorescence.
RFP sequence, based on E1010, with the number of possible pyrimidine dimer-forming sites increased to the maximum possible amount.
This is 2 copies of the CFP gene fused to each other. The duplex FP tends to be more stable, and is used in synthetic transcription factors to provide a fluorescent output of concentration as well as stability.
From David Drubin in the Silver Lab at Harvard.
amajLime, yellow-green chromoprotein (incl RBS)
GFP (mut3b) with LVA-ssrA degradation tag
GFP (mut3b) (see part BBa_E0040) with added LVA-ssrA degradation tag. The tag increases GFP turn-over rate, thus providing better temporal resolution of green fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALVA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LVA tag is reported to lead to fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations, protein stability, Km of binding to the protease, temperature [3]. References [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to GFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additonal residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (LVA in this case) are recognised by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
GFP comes from part E1010, the tag sequence was obtained from paper by Andersen et al., (1998).[2]
mVenus is a Chlamydomonas reinhardtii nuclear codon-optimized fluorescent protein. A variant of YFP, mVenus excites at 514nm and excites between 520 and 550nm. It includes two introns from RBCS2 in increase expression levels. Lauersen, Kyle., Kruse, Olaf., & Mussgnug Jan. "Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit." Applied Microbiology Biotechnology. (2015) 99;3491-3503.
This part was adapted from pOopt_mVenus_Hyg and was originally from ChlamyCollection.org
This is a device containing a promoter (BBa_J23101),a RBS (BBa_B0034), a GFP coding sequence (BBa_E0040) and two terminators (BBa_B0010, BBa_B0012) on the pSB1C3, which can be easily transformed into E.coli and produce GFP (mut3b). This is a GFP expression device, which can express GFP (mut3b) without any induction.
This is an InterLab Study required device in 2015, which was characterized by OUC-China.
T7 and GFP expression system due to T7 polymerase
Yeast optimized sfGFP N-terminus (1-214)
Green Fluorescent Protein with LVA tag for rapid degradation
Designed out of pJBA111 (Molin, 1998) with PCR to add appropriote prefix and suffix.
pJBA111, carrying GFP-LVA, was generously provided by Dr. S. Molin, The Technical University of Denmark, Lyngby Appl Environ Microbiol. 1998 Jun;64(6):2240-6. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, Molin S. PMID: 9603842
This part is composed of an arabinose-inducible promoter for superfolded GFP attached by a linker to CpxR (on the C terminus of CpxR).
mRFP derivative. Ex548nm/Em562. yeast codon optimized.
Contains N- and C-terminal linker sequences (with homology to biobrick parts BBa_E2060 (mCherry), BBa_E2020 (Cerulean CFP), and BBa_E2030 (Venus YFP) to facilitate color swapping in yeast. Adds N-"MATSG" and "GSGTA"-C. to published amino acid sequence. Double TAATAA stop codon. Missing EcoRI, HindIII, NotI, NdeI, XhoI, RsrII, BamHI, NcoI, BglI, SpeI, XbaI, and PstI. except for 5' and 3' ends no significant sequence identity runs to mCherry (BBa_E2060).
Shaner et al, 2004. Nat Biotech (22):1567-1571
pGAL1 + Flag + Brho + odr-10 + GFP + ADH1Te + pFUS1 + BFP + ADH1Te
Scissors - Primary plasmid (part 2) [tac pR, mBanana and LuxI generator]
Part of the iGEM2009 Team:Southampton rock-paper-scissors 'scissors' primary plasmid.
Regulated expression of LasI (C0178) and mBanana (K180009) by tac pR (K180000), controlled by presence of lac/IPTG or trp in the cell.
See relevant basic biobrick parts (K180000, B0034, K180009, B0015).
Made a point mutation to eliminate a PstI site in the middle and then added BioBrick ends using PCR. Sequenced using primers that bind to the pSB1A2 plasmid on either side of the brick.
mCherry from Roger Y. Tsien's lab, altered to be BioBrick compatible
P-Free JuniperGFP (Green Fluorescent Protein)
This part was created by DNA 2.0 as part of their IP-Free series of fluorescent and chromogenic proteins. It is available to use under the BioBrick Public Agreement. Excitation 508nm, Emission 521nm Molecular Weight: 26.6kDA Length: 237aa
This is the standard GFP protein reporter (E0040) cloned with the prefix/suffix required for standard 25 assembly. It can easily be used to create fusion proteins through this method.
This part contains the AgeI and NgoMIV sites as part of it's prefix/suffix that allows it to be used in standard 25 assembly. It was synthesized through PCR oligo synthesis methods using the following primers: Forward (56.88 degrees) TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCCGTAAAGGAGAAGAACTTTTC Reverse (56.95 degrees) AATACTGCAGCGGCCGCTACTAGTATTAACCGGTCTTGTCGTCATCATCTTTATAAT
This part is similar to the part BBa_E0040 commonly used as a reporter.
This part produces GFP in the presence of sigma-70 E. coli RNA polymerase; however, the promoter contains two sites for a synthetic repressor to bind. Without the presence of this repressor, the part can serve as a strong constitutive promoter, as shown in the pictures below. For more information on the repressor, visit http://2015.igem.org/Team:Pitt/Protease/Project. The GFP produced is a mutant known as GFPmut3b, and the original citation is as follows: Cormack, B.P., Valdivia, R.H., and S. Falkow. FACS-optimized mutants of green fluorescent protein (GFP). Gene 173: 33-38 (1996). (http://www.sciencedirect.com/science/article/pii/0378111995006850) It has a maximum excitation at 501 nm and maximum emission at 511 nm. For more information, see part BBa_E0040.
The part containing GFP was cut with EcoRI and XbaI, and ligated with the annealed oligos to form the completed part. Note that due to the choice of technique, a Biobrick scar site remains between the promoter and RBS.
The RBS-GFP-terminator was obtained from as part BBa_E0840 from the 2015 Distribution (Plate 2, Well 24D). The pProt promoter was obtained as two short oligonucleotides annealed to form sticky ends. The part containing GFP was then cut with EcoRI and XbaI, and ligated with the annealed oligos to form the completed part.
Tetracycline/aTc inducible promoter with GFP reporter tagged with LAA-ssrA degradation tag followed by terminator. The tagged GFP is actively degraded within cell, thus provides better temporal resolution of green fluorescence and promoter activity.
This BioBrick codes for a HBsu protein attached to a red fluorescent protein (RFP). HBsu is a non-specific DNA binding protein that binds to DNA as a homodimer. The HBsu is joined to the RFP through ten amino acid flexible linker sequence. This allows the observation of ''Bacillus subtilis'' DNA using fluorescence microscopy. The integration strategy that we opted for was to clone in the BioBrick into the Multiple cloning site of pMutin4 backbone. First we attached HindIII restriction sites on 5'end and SacII restriction sites on 3'end of the BioBrick. We then cut the pMutin4 backbone and BioBrick using the previously mentioned restriction enzymes, this allowed us to ligate the plasmid and BioBrick together. We also attached a ~300bp amyE homology region onto the 3'end of the BioBrick after the SacII to allow the single cross over and integration of the whole plasmid into the genome. The pMutin4 plasmid contains a ery+ resistance marker for ''B.subtilis'' and amp+ for ''E.coli'' and also contains lacI, lacZ and Pspac promoter which is an IPTG induced promoter which regulated the transcription of this BioBrick. An alternative method to use this part would be to clone this BioBrick out and use any Assembly protocol to attach a desired promoter, RBS and antibiotic resistance genes.
The source of the HBsu coding sequence was the ''B. subtilis'' strain 168 genome, SwissProtTM accession number: P08821. The RFP sequence was sourced from BBa_E1010 and the linker from: BBa_K105012.
pCooM RBS RFP Double terminator CooA coupled with placI+RBS
This part is formed by combination of pCooM Promoter coupled with RBS+RFP+Double terminator and CooA coupled with placI+RBS.
GFP (mut3b) with SsrA-DAS+2 degradation tag
GFP (mut3b) (see part BBa_E0040) with added engineered NYADAS-ssrA degradation tag (see part BBa_M0050). The tag increases GFP turn-over rate, thus providing better temporal resolution of green fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. SsrA tags encode peptide sequence that and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The NYADAS tag encodes peptide sequence AANDENYNYDAS is reported to have low affinity to ClpX thus its mediated degradation very much depends on the concentration of SspB (ClpX mediator).[1] The two additional residues ???NY??? extends tag between SspB and ClpX binding site, thus preventing clash when both these protein are bound to tag.[3] However, be aware that exact protein degradation rate is influenced by multiple other factors: ClpXP and ClpAP protease concentrations, protein stability, Km of binding to the protease, temperature [4]. ===References=== [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] McGinness, K. E., Baker, T. a & Sauer, R. T. Engineering controllable protein degradation. Mol. Cell 22, 701???7 (2006). [4] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to GFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additional residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (DAS in this case) are recognized by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
GFP comes from part BBa_E0040, the tag sequence was obtained from part BBa_M0051.
IP-Free TannenRFP (Red Fluorescent Protein)
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
Yellow fluorescent protein (EYFP) reporter coding sequence without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission. Annotated mutation cause a Q81L mutation that appears to not be near the active site.
BBa_E0032 yellow fluorescent protein is based on BioBrick part BBa_E0031. It has been modified to include a rapid degradation LVA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
Modified from <bb_part>BBa_E0031</bb_part>
Monomeric Red Fluorescent Protein from Discosoma striata
Mutant of the Biobrick BBa_E1010, in order to make it compatible with the Freiburg-Standard, two AgeI-Restriction sites were deleted monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm
The Biobrick has the Following Prefix and Suffix: Gatcgaattccgcggccgcttctagataaggaggagccggc Gatcctgcagcggccgctactagtattaaccggt
Venus Sequence obtained from Prof. Miyawaki. Codon optimized from Gene Design by Boeke Lab.
Gene expression for sensory rhodopsin I & II (SRI & SRII)
The part contains OmpR, which is the promoter to sense activated sensory rhodopsin I & II (SRI & SRII) after light triggering.
Strong RBS was used to allow high expression of visible fluorescence protein once the sensory rhodopsin I & II (SRI & SRII) detect the correct light spectrum.
All are constructed from previous biobricks
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with 12 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
Cellulose binding Domain from C. Fimi with const. Promotor and GFP
Cellulose binding Domain from Cellulomonas Fimi with const. Promotor J61101 and Reporter GFP
This part has two fused proteins downstream of an arabinose-inducible promoter: CpxR-IFP[1] and CpxR-IFP[2]. This construct was made in all orientations: - CpxR-IFP[1] + CpxR-IFP[2] (BBa_K1286008) - IFP[1]-CpxR + CpxR-IFP[2] (BBa_K1486010) - CpxR-IFP[1] + IFP[2]-CpxR (BBa_K1486011)
the fluorescent protein ???Keima-Red,???
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with 15 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
Lactose/IPTG inducible promoter with GFP reporter tagged with NYADAS-ssrA degradation tag followed by terminator. The tagged GFP is actively degraded within cell, thus provides better temporal resolution of green fluorescence and promoter activity.
RFP from Discosoma striata (coral) with AAV-ssrA degradation tag
Mutant RFP from Discosoma striata (coral) (see part E1010) with added AAV-ssrA degradation tag (part I11012). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYAAAV and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The AAV tag is reported to lead to reasonably fast protein degradation, degrading GFP with rate -0.012 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: 1) ClpXP and ClpAP protease and SspB mediator concentrations; 2) Protein stability; 3) Km of binding to the protease; 4) Temperature [3]. [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3]Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
RFP comes from part E1010, tag sequence was obtained from part I11012, but same tag was also usead in paper by Andersen et al., (1998).[2]
recA408 Promoter + B0030 + GFP + ssrA-tag
The promoter activated by the release of UirR (K1725420) from the transmembrane protein UirS (K1725410) in response to unidirectional UV-A light is upstream of GFP with the B0032 RBS (BBa_E5501). This allows activity in response to UV-A to be measured. This construct is expected to result in less fluorescence than K1725401. For the promoter to be activated UirR and UirS must be present and the cell has to be exposed to UV-A.
The promoter, including the BioBrick prefix and suffix, was ordered as a G-block. It was ligated to E5501 after restriction that was followed by an Azure-A-based gel extraction.
GFP - LacZ protein complex with flag tags
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, a reporter device (LacZ enzyme catalyses the reaction of the cleavage of X-Gal which results with blue color change) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the reporter. In conclusion, in this system, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system. This part contains flag tags extra from BBa_K772006 in the GFP region; because it is planned to be used in the Co-IP/SDS-PAGE assay to be isolated.
Cleavage site, LacZ and GFP sequences are gathered from Registry.
Note that there is an RBS sequence just before the protein sequence in order to eliminate the need of addition of it.
The dTomato protein is a fluorescent dimer protein that emits orange-red light when it is excited by green-yellow light. It is preferable to use ??? especially in self-made fluorometry tests ??? because the excitation wavelengths and the emission wavelengths do not overlap as much as in other fluorescent proteins. The dTomato excitation peak is at 554 nm and 50% of it is at 510 nm. The emission peak is at 581 nm and the 50% emission peak is at 629 nm.
Fluorescent proteins are useful tools for visualizing and quantifying protein expression in cells. OFP is yet another fluorescent protein, like GFP, that can be utilized as a reporter.
This part was codon-optimized for expression in E. coli. The following restriction enzyme sites were also removed: EcoRI, NotI, XbaI, SpeI, PstI, NheI, AvrII, ApoI, MfeI, NsiI, SbfI.
OFP was cloned from the tentacles of Cerianthus sp., a tube anemone. The original sequence was obtained from GenBank (VERSION AY296063.1, GI:31616578).
mCherry (BBa_J06504) compatible with Genabler assembly
mCherry (BBa_J0654) part with inward facing EarI recognition sites at the ends compatible with Genabler assembly.
To be compatible with Genabler assembly, the part has inward-facing EarI sites at the ends. When assembled by BioBrick assembly, five N-terminal residues MetAlaSerSerSer- and one C-terminal Ala residue will be added to the original sequence of BBa_E0040.
mCherry is based on the BioBrick BBa_J06504.
enhanced cyan fluorescent protein derived from A. victoria GFP
Rock-paper-scissors - Paper primary plasmid
Composite part, consisting of BBa_K180005 and BBa_K180015, but with BBa_K180005 reading along the reverse strand, and BBa_K180015 reading along the forward strand. Forms the 'paper' primary plasmid in Team:Southampton's rock-paper-scissors project design.
BBa_K180005 reading along reverse strand, and BBa_K180015 reading along the forward strand to ensure the constitutive promoter BBa_J23119 (present in BBa_K180005) has no effect over GFP or RhlI expression in BBa_K180015.
See relevant biobrick component parts (BBa_K180011 and BBa_K180012).
Tetracycline/aTc inducible promoter with GFP reporter tagged with DAS-ssrA degradation tag followed by terminator. The tagged GFP is actively degraded within cell, thus provides better temporal resolution of green fluorescence and promoter activity.Presence of SspB is crucial for effective protein degradation.
GFP, based on E0040, with overall mutation minimized
The dTomato protein is a fluorescent dimer protein that emits orange-red light when it is excited by green-yellow light. It is preferable to use ??? especially in self-made fluorometry tests ??? because the excitation wavelengths and the emission wavelengths don't overlap as much as in other fluorescent proteins. The dTomato excitation peak is at 554 nm and 50% of it is at 510 nm. Also, its emission peak is at 581 nm and its 50% emission at 629 nm (Figure 1). Characterization has been made with the Anderson promoters BBa_J23101 and (construct BBa_K1688010) and BBa_J23110 (construct BBa_K1688011).
It was derived from a monomeric mutant of the anthozoan coral RFP, DsRed
Fusion Protein of mRFP, S-layer cspB from Corynebacterium halotolerans with TAT-sequence, PT7, RBS
Fusion Protein of mRFP and S-layer cspB from Corynebacterium halotolerans with TAT-sequence, PT7 and RBS
Gibson Assembly of BBa_E1010 and BBa_K525224
This part encodes a reporter cassette for the Cre Recombinase (e.g parts XYZ) in Yeast. An active Cre protein will switch expression from RFP to NanoLuc luciferase.
Constitutive promoter+37℃ induced RBS+tetR+double terminator+Ptet+RBS+GFP+double terminator
We want to utilize a temperature-regulated mechanism to control the expression period of a crystal protein. Therefore, we took advantage of the interaction between 37?? C induced RBS, tetR gene and pTet promoter to create the temperature regulating circuit. The following is a description of our prescribed strategy. First, we use the constitutive promoter, 37?? C induced RBS (K115002), the tetR gene and terminators. We will call this part the ???Regulator???.
This is a device containing a promoter (BBa_J23117),a RBS (BBa_B0034), a GFP coding sequence (BBa_E0040) and two terminators (BBa_B0010, BBa_B0012) on the pSB1C3, which can be easily transformed into E.coli and produce GFP (mut3b). This is a GFP expression device, which can express GFP (mut3b) without any induction.
This is one of 2015 InterLab Study required devices. The strength of the promoter is weak.
Lactose/IPTG inducible promoter with GFP reporter tagged with AAV-ssrA degradation tag followed by terminator. The tagged GFP is actively degraded within cell, thus provides better temporal resolution of green fluorescence and promoter activity.
Modificaitons to Clontech EYFP by Reshma Shetty
Superfolder GFP under control of strong constitutive promoter and RyhB siRNA domain
Fusion Protein of S-Layer SgsE and mCitrine
Fusion Protein of BBa_K525303 and BBa_J18931
This is a construct for testing promoter strength by measuring RFP fluorescence.
pCopA MymT sfGFP with plasmid constitutively expressed CueR
ECFP, yeast optimized for fusion proteins
Our BioBrick is cyan fluorescent protein which can be used to label proteins in yeast. Therefor it has specific characteristics of BioBrick part for fusion proteins (see design details).
To enable the fusion with other domains in frame the vector of this BioBrick has no base pair in between the restriction side and the BioBrick. Furthermore, this coding sequence does not include a start codon.<br\n> For more information about this issus, see:<br\n> Phillips, I.E. and Silver, P.A. "A new Biobrick Assembly Strategy Designed for Facile Protein Engineering." <br\n> DSpace http://hdl.handle.net/1721.1/32535 (2006).
We changed the format of the ECFP coding part of BBa_E2020 into Phillips/Silver standard.
Rock - Primary plasmid (part 2) [tac pR, mCherry and LasI generator]
Part of the iGEM2009 Team:Southampton rock-paper-scissors 'rock' primary plasmid.
Regulated expression of LasI (C0178) and mCherry (K180008) by tac pR (K180000), controlled by presence of lac/IPTG or trp in the cell.
See relevant basic biobrick parts (K180000, B0034, K180008, B0015)
Alkane Selector for intracellular alkane production
RFP from Discosoma striata (coral) with LAA-ssrA degradation tag (wt)
Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LAA-ssrA degradation tag (part BBa_M0050). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is E. coli wild type ssrA tag and is reported to lead to very fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; Protein stability; Km of binding to the protease; Temperature [3]. Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LAA-ssrA degradation tag (part BBa_M0050). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is E. coli wild type ssrA tag and is reported to lead to very fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; Protein stability; Km of binding to the protease; Temperature [3]. Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LAA-ssrA degradation tag (part BBa_M0050). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is E. coli wild type ssrA tag and is reported to lead to very fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; Protein stability; Km of binding to the protease; Temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012)
RFP comes from part BBa_E1010, tag sequence was obtained from part BBa_M0050, but same tag was also used in paper by Andersen et al., (1998).[2]
SuperYFP2 (BBa_K864100) with 6x His tag for protein purification.
XylR coding device-GFP coding device with Pu
XylR coding device (BBa_K2023002) and GFP coding device with Pu (BBa_K2023016)
Green fluorescent protein (GFP) with N-terminal His-Tag
This part is a direct fusion of the theophylline riboswitch (BBa_K784005) and the mCherry protein (BBa_J06504) which was amplified from BBa_J06702. The part was generated by [http://openwetware.org/wiki/Assembly_pcr Assembly_pcr]. This part was to be used in order to characterize the effect of theophylline concentration on the level of translation of the mCherry protein.
The theophylline riboswitch had to be directly fused to the start codon of the mCherry protein. To achieve this, [http://openwetware.org/wiki/Assembly_pcr Assembly_pcr] was used.
This part is a composite part of BBa_K784005 and BBa_J06504
Yeast Optimized sfGFP-C + ADH1 terminator + CaUra3 Cassette
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 6 AA linker as spacer (BBa_K1680000). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
Constitutive promoter (J23101) and GFP (K1819000)
Constitutive promotor from Anderson library (BBa_J23101) assembled to green fluorescent protein attached to linker (BBa_K1819000).
This part results from the assembly of Bba_J23101 and BBa_K1819000
This part has a fused protein downstream of an arabinose-inducible promoter: CpxR-IFP[2]. Biobrick BBa_K1486012 has CpxR-IFP[1] with an arabinose promoter. This construct was made in both orientations: BBa_K1486015 is IFP[2]-CpxR (with an arabinose promoter).
Both orientations were made, as well as for IFP[1] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put before each one of them.
mCherry fluorescent protein - Rights owned by Clontech. Sequence is commercially available from Clontech. Sequence obtained through PCR amplification, with BioBrick prefix and suffix added through in-house designed primers.
PstI site within the mCherry sequence - has to be assembled using BioBrick assembly in such an order that any successive digestions will not require PstI, otherwise the mCherry sequence will be cut as well as the site of interest.
GFP (mut3b) with DAS-ssrA degradation tag
GFP (mut3b) (see part BBa_E0040) with added DAS-ssrA degradation tag (see part BBa_M0052). The tag increases GFP turn-over rate, thus providing better temporal resolution of green fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. SsrA tags encode peptide sequence that is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The DAS tag encodes peptide sequence AANDENYADAS is reported to have low affinity to ClpX thus its mediated degradation very much depends on the concentration of SspB (ClpX mediator).[1] However, be aware that exact protein degradation rate is influenced by multiple other factors: ClpXP and ClpAP protease concentrations, protein stability, Km of binding to the protease, temperature [3]. ===References=== [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
GFP comes from part BBa_E0040, the tag sequence was obtained from part BBa_M0052.
Monomeric red fluorescent protein (mRFP1) is a derivative of the red fluorescent protein cloned from Discosoma coral (DsRed) (1). The successful use of mRFP1 in Aspergillus nidulans has previously been described (2).
This part was designed for the Plug'n'Play with DNA assembly standard (RFC 80), therefore this part contains tails at the 5' and 3' ends as shown on the figure below. Please notice that mRFP1-module is not the same as BBa_K678031 or BBa_K678032.
Fusion Protein of S-Layer SbpA and mCitrine
Fusion Protein of BBa_K525403 and BBa_J18931
rbs-GFP(LVA)-terminators (Backbone: pSB1A2)
This part is a generator that lacks a promoter. The GFP has a LVA degradation tail. Upon expression, the GFP will give green fluorescence color.
The repression of heat shock gene expression (ROSE) element is an RNA element found in the 5' UTR of some heat shock protein's mRNAs. The ROSE element is an RNA thermometer that negatively regulates heat shock gene expression. The secondary structure is thought to be altered by temperature, thus it is an RNA thermometer. This structure blocks access to the ribosome binding site at normal temperatures. During heat shock however, the structure changes freeing the ribosome binding site and allowing expression to occur. RNA Thermometers are the only known single-component regulators of gene expression.(1) RNA Thermometers are thermosensors that regulate gene expression by temperature-induced changes in RNA conformation. Naturally occurring RNA Thermometers exhibit complex secondary structures which are believed to undergo a series of gradual structural changes in response to temperature shifts.(2) RNA Thermometers often regulate genes required during either a heat shock or cold shock response, but have been implicated in other regulatory roles such as in pathogenecity and starvation.(3)
Bradyrhizobium japonicum USDA 110 and Registry
The protein comes from Aequorea victoria. This jellyfish uses GFP (Green Fluorescent Protein) in order to convert the blue luminescence emitted by the aequorine into a green luminescence
The first two nucleotides of the GFP start codon were removed to create this fusion brick.
The GFP fusion brick was derived from the GFP BioBrick, [http://partsregistry.org/Part:BBa_E0040 BBa_E0040].
mBanana fluorescent protein - Rights owned by Clontech. Sequence is commercially available from Clontech. Sequence obtained through PCR amplification, with BioBrick prefix and suffix added through in-house designed primers.
PstI site within the mBanana sequence - has to be assembled using BioBrick assembly in such an order that any successive digestions will not require PstI, otherwise the mCherry sequence will be cut as well as the site of interest. Alternatively, the assembly can be performed using non-standard BioBrick assembly. Part cannot be submitted to registry as the rights are owned by Clontech. The mCherry sequence is however commercially available from Clontech in the pmBanana vector.
Inducible Green Fluorescent Protein UnaG+6xHis-tag
A CDS of Dronpa protein with a mutation in the betha-strand 7 in Lys145 for Asn.
Pectinidae sp. sequence found in the following article Lummer, M., Humpert, F., Steuwe, C., Caesar, K., Sch??ttpelz, M., Sauer, M. and Staiger, D. (2011), Reversible Photoswitchable DRONPA-s Monitors Nucleocytoplasmic Transport of an RNA-Binding Protein in Transgenic Plants. Traffic, 12: 693???702. doi: 10.1111/j.1600-0854.2011.01180.x
This is the tightly regulated lac operon BBa_K389050 with a mRFP gene as readout.
lacIq integrated in promoter so good regulation, mRFP as readout
Constitutive promoter (J23117) and GFP (S05308)
Constitutive promotor from Anderson library (BBa_J23117) assembled to green fluorescent protein attached to linker (BBa_S05308). This part was used to characterize BBa_K1819000 and compare its efficiency to green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (BBa_E0040).
This part results from the assembly of BBa_J23117 and BBa_S05308
N-terminal part of the Split-flourophore YFP, fused to the trans-membrane region of the Erbb1/EGF-receptor.
Derived by cloning the parts BBa_K157002, BBa_K157008. For further information see the description of the parts mentioned above.
Rock-paper-scissors - Rock primary plasmid
Composite part, consisting of BBa_K180011 and BBa_K180012, but with BBa_K180011 reading along reverse strand, and BBa_K180012 reading along the forward strand. Forms the 'rock' primary plasmid in Team:Southampton's rock-paper-scissors project design.
This part has been specified as a basic BioBrick, due to some of the component parts reading in reverse (unable to specify using the component BioBrick format).
fwYellow, yellow chromoprotein (incl RBS, J23106)
[http://partsregistry.org/Part:BBa_I13522 BBa_I13522] with His-tag and without the terminators
Lactose/IPTG inducible promoter with RBS, RFP reporter tagged with LVA-ssrA degradation tag and terminator. The tagged RFP is actively degraded within cell, thus provides better temporal resolution of red fluorescence and promoter activity. This part can be considered as improved version of BBa_J04451 as it does not contain StuI restriction site coding for additional 'RP' residues between RFP and LVA tag.
This part has a fused protein downstream of an arabinose-inducible promoter: IFP[1]-CpxR. Biobrick BBa_K1486015 has IFP[2]-CpxR with an arabinose promoter. This construct was made in both orientations: BBa_K1486012 is CpxR-IFP[1] (with an arabinose promoter).
Both orientations were made, as well as for IFP[2] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put between the promoter and the cds.
This part was designed for the Plug'n'Play with DNA assembly standard (RFC 80), therefore this part contains tails at the 5' and 3' ends as shown on the figure below. Please notice that mRFP1-TS is not the same as BBa_K678030 or BBa_K678031.
This part has a fused protein downstream of an arabinose-inducible promoter: IFP[2]-CpxR. Biobrick BBa_K1486014 has IFP[1]-CpxR with an arabinose promoter. This construct was made in both orientations: BBa_K1486013 is CpxR-IFP[2] (with an arabinose promoter).
Protein coding sequence for a mutant (K145N) of the fluorescent protein Dronpa, codon optimized for Yeast. The mutation leads to photoswitchable monomerisation/multimerisation behaviour (as described by Zhou et al. 2012).
As a BD part, the stop codon is included.
mOrange, Yeast optimized for fusion proteins
Our BioBrick is red-orange fluorescent protein which can be used to label proteins in yeast. Therefor it has specific characteristics of a BioBrick part for fusion proteins (see design details). <br\n>
We changed the format of BBa_E2050 into Phillips/Silver standard.
With this system it is possible to measure the activity of a virA receptor and the vir promoter, respectively. The reporter is the mRFP from the partsregistry.
medium strong constitutive promotor, terminator to keep basal expression of mRFP low
partsregistry, synthetic gene by Mr. Gene, TI-plasmid from ''Agrobacterium tumefaciens'' C58
Part is composed of TIR domain of Traf6 and GFP linked with 6 aminoacids long linker. GFP serves as reporter. For usage in eukaryotes.
A SV40 nuclear localization signal was added to the phototoxic protein KillerRed.
Constitutive (tetR repressible) LacI and RFP expression
This part constitutively expresses LacI and RFP except when repressed by TetR. When cells containing this part are exposed to the inducer molecule IPTG, it can be used to regulate lac-repressible promoters. It was designed to function as an IPTG-controllable tuner for the [http://partsregistry.org/Part:BBa_K546546 Double Tunable Synchronized Oscillator]. It is advisable to operate this part on a low or medium copy plasmid.
GFP (mut3b) with LAA-ssrA degradation tag
GFP (mut3b) (see part BBa_E0040) with added LAA-ssrA degradation tag (see part BBa_M0050). The tag increases GFP turn-over rate, thus providing better temporal resolution of green fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes E.coli wild type ssrA peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is reported to lead to fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations, protein stability, Km of binding to the protease, temperature [3]. ===References=== [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to GFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additional residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (LAA in this case) are recognized by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
GFP comes from part BBa_E1010, the tag sequence was obtained from part BBa_M0050.
GFP targeted to the peroxisomes of Aspergillus nidulans
RBS+GFP for expression in Bacillus subtilis
It is succesfully joined B.subtilis RBS with GFP reporter protein. Can be used to measure strenght of B.subtilis promoters. In order to work with it you must first add appropriate terminator at the end. We recommend BBa_K780000 - a terminator designed for use with B.subtilis.
RBS was taken from 'host-takover module' from B.subtilis Bacteriophage SPO1 and GPF is derived from jellyfish Aequeora victoria (BBa_E0040).
This part accepts POPS to produce a single mRNA containing EYFP and ECFP under the strong clonetech RBS
SBFP2 (strongly enhanced blue fluorescent protein)
This part codes for orange fluorescent protein (OFP). Fluorescent proteins are useful tools for visualizing and quantifying protein expression in cells. OFP is yet another fluorescent protein, like GFP, that can be utilized as a reporter.
This part was codon-optimized for expression in ''E. coli''. The following restriction enzyme sites were also removed: EcoRI, NotI, XbaI, SpeI, PstI, NheI, AvrII, ApoI, MfeI, NsiI, SbfI.
OFP was cloned from the tentacles of ''Cerianthus'' sp., a tube anemone. The original sequence was obtained from GenBank (VERSION AY296063.1, GI:31616578).
This device acts as a detector for glucose (and certain derivatives of it). The double transcription terminator ensures, that only the gene downstream of the promotor zwf is influenced by it. The zwf promotor (BBa_K1840002) is contained in the genome of Pseudomonas putida and is situated in front of the enzyme zwf (glucose 6-phosphate dehydrogenase). The repressor HexR can bind the zwf promotor sequence and thus prevent the transcription of the zwf gene. In case the glucose derivative 2-Keto-3-deoxy-6-phosphogluconate is present, it binds to the promotor and leads to a subsequent conformational change. Hence, the repressor can no longer bind and the gene(s) downstream can be transcribed. In our device, the downstream gene is mCherry, codon optimized for Pseudomonas (BBa_K1840004). Therefore, in the presence of glucose, mCherry is expressed. In various experiments, we could see that this device is in deed working.
As promotor sequence, we assumed all DNA in front of the zwf gene up to the next gene, coding for the repressor HexR. Since it is encoded on the opposite strand, there is the possibility, that zwf functions as a promotor for this gene as well. Therefore the double terminator was needed.
The double terminator was already registered in the iGEM catalogue. The zwf promotor comes from the genome of Pseudomonas putida. The mCherry is codon optimized for expression in Pseudomonas.
mCherry, Yeast optimized for fusion proteins
Our BioBrick is red fluorescent protein which can be used to label proteins in yeast. Therefor it has specific characteristics of a BioBrick part for fusion proteins (see design details). <br\n>
We changed the format of BBa_E2060 into Phillips/Silver standard.
This is a device that contain promoter BBa_J23117, RiboJ and BBa_I13504.
We want to test the strength of the J23117 without disturbance from genetic context.
GFP - LasR anchor-inducer protein complex
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, an inducer regulatory protein (LasR is responsible to induce pLas promoter in the presence of 6HSLO3 which is a chemical compound sythesized by an enzyme that LasI gene codes.) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the reporter. In conclusion, in this system, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system.
Cleavage site, LasR and GFP sequences are gathered from Registry.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (His-tag)
Fusion Protein of S-Layer SbpA and mCherry RFP
Fusion Protein of BBa_K525403 and BBa_J18932
enhanced version of ECFP, yeast-optimized
Contains N- and C-terminal linker sequences (with homology to biobrick parts BBa_E2060 (mCherry), BBa_E2050 (mOrange), and BBa_E2030 (Venus YFP) to facilitate color swapping in yeast. Adds N-"MATSG" and "GSGTA"-C. to published amino acid sequence. Inserts a "V" after normal start M. Double TAATAA stop codon. Missing EcoRI, HindIII, NotI, NdeI, XhoI, RsrII, BamHI, NcoI, BglI, SpeI, XbaI, and PstI. Except for 5' and 3' ends no significant sequence identity runs with Venus YFP (BBa_E2030) or to GFP S65T as found in O'Shea deletion strain collection (originally from plasmid pFA6-GFP(S65T)-His3MX6).
Rizzo MA, et al. (2004) Nat Biot (22):445-9
This part accepts POPS to produce ECFP under the weak RBS B0031
The second portion of the RFP gene, after the HixC insertion point.
This part accepts POPS to generate EYFP under the medium strength RBS B0032.
==Part Description== This is a composite part made from Green Fluorescent Protein (GFP) and a double terminator. Green Fluorescent protein (originally from part [http://partsregistry.org/Part:BBa_E0040 BBa_E0040]) that will glow green when excited by the correct wavelengths and thus can be used as a reporter gene. This part is directly fused to a terminator (originally from part [http://partsregistry.org/Part:BBa_B0014 BBa_B0014]) so it is ideal to be fused at the end of your construct. ==Table of Statistics== {| style="color:white; background-color:#0066CC;" cellpadding="20" cellspacing="0" border="2" !Chassis !Monomer/Multimer !Aminoacid Length/Weight !Localisation !Structure !Excitation wavelengths !Emmision wavelengths |- |Prokaryotic and Eukaryotic |Bipartite |274 aminoacids | |beta-barrel(for GFP) hairpins for Terminator |501nm |511nm |} ==References== #Valdivia pmid=8707053
No additional design considerations. The parts were fused together following RFC10 assembly.
GFP originates from jellyfish Aequeora victoria. For more details visit the [http://www.expasy.ch/uniprot/P42212 SwissProt entry]. The double terminator was designed by Reshma Shetty. For more details visis the [http://partsregistry.org/Part:BBa_B0014 original part page].
This is N-terminal of mCherry, that can be used in Bimolecular Fluorescence Complementation system (BiFC).
PartsRegistry J06505, originally from Discosoma sp.
GFP generator. Inducible using IPTG/Lactose.
Repression of heat shock gene expression (ROSE) element The repression of heat shock gene expression (ROSE) element is an RNA element found in the 5' UTR of some heat shock protein's mRNAs. The ROSE element is an RNA thermometer that negatively regulates heat shock gene expression. The secondary structure is thought to be altered by temperature, thus it is an RNA thermometer. This structure blocks access to the ribosome binding site at normal temperatures. During heat shock however, the structure changes freeing the ribosome binding site and allowing expression to occur.[2][3] A partial structure of this RNA element has been determined using NMR.[1] ROSE1 and ROSEAT2 are specific examples of ROSE elements.[4] ROSE1 is found in Bradyrhizobium japonicum whereas ROSEAT2 is a closely related element from Agrobacterium tumefaciens. The two RNA elements have similar secondary structures with ROSE1 having an extra hairpin.[4] All ROSE elements contain a characteristic 'bulged G' opposite the Shine-Dalgarno sequence binding site, without this nucleotide the RNA thermometer loses its temperature-sensitivity.[5]
Super Yellow Fluorescent Protein 2 (SYFP2)
This part codes for the bright yellow fluorescent protein SYFP2. SYFP2 is a GFP based monomeric protein with a narrow fluorescence emission spectrum with a maximum at 515 nm. Bacteria expressing SYFP2 are reported to be 12 times brighter than those expressing EYFP(Q69K) and almost 2-fold brighter than bacteria expressing Venus (Ref. 10.1021/bi0516273). Codon optimized for expression in E coli by DNA 2.0. Mutations compared to wtGFP amino acid sequence (GenBank Accession number M62653) are F46L F64L S65G S72A M153T V163A S175G T203Y A206K.
Amino acid sequence taken from the article by Kremers et al 2006.
slr2016 signal sequence + GFP fusion for secretion of GFP
Ligation of the slr2016 and GFP fusion biobricks.
GFP had to be a fusion brick so that translation would continue in frame.
''slr2016'' signal sequence was derived from ''Synechocystis'' sp. PCC 6803 while GFP fusion was modified from the BioBrick part BBa_E0040.
GFP - LasR protein complex with flag tags
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, an inducer regulatory protein (LasR is responsible to induce pLas promoter in the presence of 6HSLO3 which is a chemical compound sythesized by an enzyme that LasI gene codes.) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the reporter. In conclusion, in this system, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system. This part contains flag tags extra from BBa_K772006 in the GFP region; because it is planned to be used in the Co-IP/SDS-PAGE assay to be isolated.
Super Yellow Fluorescent Protein 2 (SYFP2) in RFC[25]
Improved part of BBa_K864100 which originally cannot be fused to other protein domains. This improved version is flanked by RFC[25] pre- and suffix for further protein fusions.
mRFP (DsRed) derived, Ex587/Em610, yeast codon optimized
Codon optimized for yeast. Contains N- and C-terminal linker sequences (with homology to biobrick parts BBa_E2050 (mOrange), BBa_E2020 (Cerulean CFP), and BBa_E2030 (Venus YFP) to facilitate color swapping in yeast. Adds N-"MATSG" and "GSGTA"-C. to published amino acid sequence. Double TAATAA stop codon. Missing EcoRI, HindIII, NotI, NdeI, XhoI, RsrII, BamHI, NcoI, BglI, SpeI, XbaI, and PstI. except for 5' and 3' ends no significant sequence identity runs to mOrange (BBa_E2050).
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with 9 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
This part has a fused protein downstream of an arabinose-inducible promoter: CpxR-IFP[1]. Biobrick BBa_K1486013 has CpxR-IFP[2] with an arabinose promoter. This construct was made in both orientations: BBa_K1486014 is IFP[1]-CpxR (with an arabinose promoter).
Both orientations were made, as well as for IFP[2] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put before each one of them.
Lux pI+RBS+LuxR+2TM+Lux pR+RBS+LuxI+2TM+Lux pR+RBS+GFP+2TM+Lux pR+RBS+α-ALS+2TM
Junjie zhang, Yichong wang, Zewei yan, Rui chen and Xuenian chen
BBa_K1072007 is a composite divece, including LuxR, LuxI, GFP and α-ALS genes. LuxR is under control of a constitutive promotor, Lux pI. Produce, LuxR, forms a complex with the quorum sensing molecule AHL and this complex subsequently increases the transcriptional rate of the lux pR promoter while also decreas the transcriptional rate of the lux pL promoter. The other genes are under control of this lux pR promoter. The generator, Lux pR+RBS+LuxI+B0015, express AHL which will combine with LuxR. GFP also is under control of Lux pR, presenting as reporter. α-ALS，as our desire produce, will transcrip activated by LuxR and AHL complex. If co-transforming with B0015 , this system will transcript α-ALS periodically. Finally, this part just is the combination of BBa_K1072000 and BBa_K1072006 .
Some part get from registry and α-ALS gene originate from Lactococcus lactis, respectively.
GFP - LacZ anchor-inducer protein complex
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, a reporter/inducer device or protein (enzyme etc.) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the inducer/reporter. In conclusion, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system.
RFP from Discosoma striata (coral) with LVA-ssrA degradation tag
Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LVA-ssrA degradation tag. The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. This tag is commonly attached to repressor proteins for use in various gene networks (e.g., oscillators). The tag encodes peptide sequence AANDENYALVA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LVA tag is reported to lead to fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; protein stability; Km of binding to the protease; temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to RFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additonal residues and restriction sites. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (LVA in this case) are recognised by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
RFP comes from part BBa_E1010, tag sequence was obtained from paper by Andersen et al., (1998).[2]
Tetracycline/aTc inducible promoter with GFP reporter tagged with AAV-ssrA degradation tag followed by terminator. The tagged GFP is actively degraded within cell, thus provides better temporal resolution of green fluorescence and promoter activity.
This is a RFC12 compatible part based off of BBa_E0040.
Amplified via colony PCR directly from the BBa_E0040 cell stock. Forward Primer: 5' GTTTCTT C GAATTC GCGGCGC T ACTAGT CGTAAAGGAGAAGAACTTTTCACTG 3' Reverse Primer: 3' CACATGGCATGGATGAACTATACAAA 5' so 5' GTTTCTT C CTGCAG CGGCCGC T GCTAGC TTTGTATAGTTCATCCATGCCATGTG 3'
Derived by cloning of the parts BBa_K157001, BBa_K157004, BBa_K157013, BBa_K157002, BBa_K157009 and BBa_K157007. For further information see the description of the parts mentioned above.
Theophylline Responsive Riboswitch 1G1 with Engineered RBS+GFP generator
Siyang HAO, Tong MU, Zheng LUO, Mengqi ZHANG
This is a GFP generator with a theophylline responsive riboswitch sequence. The mRNA with a theophylline riboswitch in it would respond to theophylline concerntration, producing different amount of GFP. Under the control of different promoters, it could be used to demonstrate the theophylline responsive curve, providing data for modeling. The theophylline riboswitch sequence could also be used to regulate the translation of other genes.
The RBS of this device is altered into AGGAGGU. When changing the RBS, the anti-RBS sequence should be changed as well.
Theophylline riboswich is composed of a synthesized theophylline aptamer and a regulatory platform.
Theophylline responsive riboswitch Theo 1G1
IP-Free EiraCFP (Cyan Fluorescent Protein)
This part was created by DNA 2.0 as part of their IP-Free series of fluorescent and chromogenic proteins. It is available to use under the BioBrick Public Agreement. Excitation 398nm, Emission 498nm Molecular Weight: 26.0kDA, Length: 233 amino acids
DNA 2.0 have created a large set of new colored and fluorescent proteins by back-translating a set of sequences from Genbank, using their proprietary GeneDesigner software. To create these proteins, they simultaneously minimized sequence differences and and derived an E. coli codon bias for high protein expression. Gene fragments were designed to create chimeric genes when pooled. A subset of the resultant chimeras were selected as base genes from which multiple iterations of libraries with targeted substitutions were made. (from DNA 2.0)
From Pectinidae sp. It is a CDS of green fluorescent protein Dronpa.
This part produces GFP in the presence of T7 RNA polymerase. It contains a T7 promoter, a strong RBS, the coding region for GFP, and a double terminator. The GFP is a mutant known as GFPmut3b, and the original citation is as follows: Cormack, B.P., Valdivia, R.H., and S. Falkow. FACS-optimized mutants of green fluorescent protein (GFP). Gene 173: 33-38 (1996). (http://www.sciencedirect.com/science/article/pii/0378111995006850) It has a maximum excitation at 501 nm and maximum emission at 511 nm. For more information, see part BBa_E0040
The RBS-GFP-terminator was obtained from as part BBa_E0840 from the 2015 Distribution (Plate 2, Well 24D). The T7 promoter was obtained as two short oligonucleotides annealed to form sticky ends. The part containing GFP was then cut with EcoRI and XbaI, and ligated with the annealed oligos to form the completed part. Note that due to the choice of technique, a Biobrick scar site remains between the promoter and RBS.
cI coding protein on plasmid backbone J61002
This BioBrick codes for a HBsu protein attached to a superfolded green fluorescent protein (sfGFP). HBsu is a non-specific DNA binding protein that binds to DNA as a homodimer. The HBsu is joined to the sfGFP through ten amino acid flexible linker sequence. This allows the observation of ''Bacillus subtilis'' DNA using fluorescence microscopy. The integration strategy that we opted for was to clone in the BioBrick into the Multiple cloning site of pMutin4 backbone. First we attached HindIII restriction sites on 5'end and SacII restriction sites on 3'end of the BioBrick. We then cut the pMutin4 backbone and BioBrick using the previously mentioned restriction enzymes, this allowed us to ligate the plasmid and BioBrick together. We also attached a ~300bp amyE homology region onto the 3'end of the BioBrick after the SacII to allow the single cross over and integration of the whole plasmid into the genome. The pMutin4 plasmid contains a ery+ resistance marker for ''B.subtilis'' and amp+ for ''E.coli'' and also contains lacI, lacZ and Pspac promoter which is an IPTG induced promoter which regulated the transcription of this BioBrick. An alternative method to use this part would be to clone this BioBrick out and use any Assembly protocol to attach a desired promoter, RBS and antibiotic resistance genes.
The source of the HBsu coding sequence was the ''B. subtilis'' strain 168 genome, SwissProtTM accession number: P08821. The sfGFP sequence was sourced from BBa_E0040 and the linker from: BBa_K105012.
GoldenGate compatible Green Flourescent Protein (GFP)
A variation of the part BBa_E0040 that has been made compatible for Golden Gate by removal of internal BsaI and BpiI recognition sequences. Fluoresces under UV light when the gene is expressed.
Domestication for GoldenGate by removal of internal BsaI and BpiI recognition sequences.
C-terminal fragment (Amino acids157-237) of the monomeric[5] yellow fluorescent protein variant ???Venus??? [4], designed for fusion/BiFC[1,3]. Reassembly with Part Bba_K157008 (Split-Venus-nYFP) generates the complete, working YFP ???Venus???, whereas combination with the n-terminal fragment (Part Bba_K157006) of the monomeric cyan fluorescent Protein ???CeruleanR206K??? (Part Bba_I757009) results in green fluorescence[2]. - [1] Chang-Deng Hu, Yurii Chinenov, Tom K. Kerppola: ???Visualization of Interactions among bZIP and Rel Family Proteins in Living Cells Using Bimolecular Fluorescence Complementation???, Molecular Cell, Vol. 9, 789???798, April, 2002 - [2] Chang Deng Hu, Tom K. Kerppola: ???Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis???, Nat Biotechnol. 2003 May; 21(5):539-545 (doi:10. 1038/nbt816) -[3] Tom K. Kerppola: ???Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells???, Nat Protoc. 2006;1(3):1278-1286 (doi:10.1038/nprot.2006.201) -[4]Nagai, T. et al. ???A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications??? J. Biol. Chem. 276, 29188-29194, 2001 -[5]Roger Y. Tsien et al. ???Creating new fluorescent probes for cell biology???, Nature Biotechnology Reviews, Vol. 3, 906-918, 2002
Enhanced Cyan Fluorescent Protein (eCFP) in RFC[25]
This Biobricks is a improved version BBa_E0020 which codes for the Enhanced Cyan Fluorescent Protein. We improve the function of the biobrick, to allow protein fusions which is originally not possible due to RFC[10] pre- and suffix. This part is flanked by RFC[25] pre- and suffix for further protein fusions.
CBD of C. cellulovorans cellulose binding protein gene with const. Promoter (J23100+J61101) and GFP
Cellulose binding Domain of Clostridium cellulovorans [http://www.ncbi.nlm.nih.gov/nuccore/M73817 cellulose binding protein gene (cbp A)] with a constitutive promoter and RBS ( J61101 ) and a fused-GFP protein with His-tag.
Was made to switch from a T7-promoter to a constitutive promoter
Cellulose binding domain form a cloning-vector. J61101 and [http://partsregistry.org/Part:BBa_I13522 GFP] from the partsregistry.
Superfolder Cyan Fluorescent Protein is a derivative of superfolder Green Fluorescent protein. Compared to CFP it displays improved stability and strong cyan fluorescence.
PartsRegistry BBa_I746909, originally come from Aequorea victoria
This is the ompC promoter, controlled by the response regulator OmpR and sensor kinase EnvZ, joined to Enhanced Yellow Fluorescent Protein.
Made by joining ompC promoter BioBrick BBa_R0082 to a PCR product including RBS BBa_B0034 and EYFP BBa_E0030.
CFP under the control of a CMV promoter.
This part was split from its plasmid backbone BBa_K157040.
Cyan fluorescent protein (ECFP) reporter coding sequence without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission.
BBa_E0022 cyan fluorescent protein is based on BioBrick part BBa_E0021. It has been modified to include a rapid degradation LVA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
Modified from <bb_part>BBa_E0021</bb_part>.
we REMOVED the extra base from part BBa_I715020(easier to fuse with other proteins) when designing PCR primers to make the protein fusion easier.
This part is gained from part BBa_I715020.
PAmCherry (photoactivatable red fluorescent protein)
PAmCHERRY is a photoactivatable red fluorescent protein. PAmCherry fluoresces when exposed to 350???400 nm light while does not fluoresce at other wavelengths. It is used a reporter tag: by expressing in cells or localizing to sub-cellular locations, one can gain spatial information within the cell by track organelles, proteins, and the whole cell itself. One can visualize the photoactivated PAmCherry with the same filter sets used for other red fluorescent proteins.
Codon optimization for yeast and internal restriction site removal to be compatible with our Golden Gate cloning scheme.
Synthesized in house. Non-genomic DNA sequence. Mutated version of RFP.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212)
GFP-AAV [Pst1 site in earlier version knocked out]
pTet + 37 oC RNA thermometer + mCherry (LVA)
This part contains a promoter regulated by TetR protein. The Transcription is regulated by a 37 oC riboswitch. The protein codified is a RFP with a LVA tag.
The prefix sites are EcoRI and XbaI and the sufix sites are SpeI and PstI.
Obtained synthetically. Promoter pTet. Based on a fusion of a RBS and a Riboswitch. RFP with a LVA tag (Further information coming soon).
iGEM 2012 Grenoble Team proposes a new design and application to the pBAD promoter(paraBAD).
In order to create a biological AND gate the 2012 Grenoble team worked on the cAMP-CRP complex and arabinose inductible promoter. The activity of the promoter was tested under several conditions (http://2012.igem.org/Team:Grenoble/Biology/Protocols/AND_test)using a GFP as a reporter (on a pUA66). We tested this activity in BW25113 ΔcyaA in different culture media (Glucose + Acetate; Glycerol; Acetate).
Design note: http://www.weizmann.ac.il/mcb/UriAlon/Papers/Zaslaver_Ecoli_library.pdf
The part comes from the plasmid collection Alon (http://www.weizmann.ac.il/mcb/UriAlon/Papers/Zaslaver_Ecoli_library.pdf). However, pBAD is a natural promoter in E. Coli. It regulates the production of three proteins (AraA, AraB and AraD) which form the arabinose operon. Their production enables the use of arabinose as a carbon source. It has six regulation sites, five of them are devoted to AraC fixation (three are repressing, one has a dual activity and one is an activator); the last one is a CRP binding site (positive activity). This promoter is activated when both activated CRP and AraC are bind to it. GFP comes from the protein comes from Aequorea victoria. This jellyfish uses GFP (Green Fluorescent Protein) in order to convert the blue luminescence emitted by the aequorine into a green luminescence. Aequorea victoria: is a jellyfish that can be found off the coast of north America.
Paper - Primary plasmid (part 2) [tac pR, GFP and RhlI generator]
Regulated expression of RhlI (C0170) and GFP (E0040) by tac pR (K180000), controlled by presence of lac/IPTG or trp in the cell.
See relevant basic biobrick parts (K180000, B0034, E0040, B0015).
Since this is an AB part, there is an ATG but no stop codon. For more information on AarI cloning, please refer to the UCSF 2008 igem page.
GFP - LacZ protein generator is generated with the aim of inducing a reporting reaction aftermath of the activation of EpCAM detection system. (BBa_K772100) For more information: BBa_K772006
To achieve a constitutive production, pTetR promoter is added in the upstream region of the original part.
This plasmid contains code for pBAD promoter, AIP receiver Agr A and Agr C, GFP along with double terminator.
Sequence was cloned using BBa_E0240 and BBa_I746103 from iGEM parts registry.
This part consists of a fusion protein Lpp-rbs-protein under the pLac promoter. The Lpp is a signal peptide. The ompA domain of the fusion directs the protein to the surface membrane. The pLac promoter can be repressed by LacI protein. This part lacks a terminator.
The pLac promoter, rbs sequence are obtained from the registry The fusion protein coding sequence is self-designed
mTagBFP (BBa_K592100) compatible with Genabler assembly
mTagBFP (BBa_K592100) part with inward facing EarI recognition sites at the ends. It is compatible with Genabler assembly.
To be compatible with Genabler assembly, the part has inward-facing EarI sites at the ends. When assembled by BioBrick assembly, five N-terminal residues MetAlaSerSerSer- and one C-terminal Ala residue will be added to the original sequence of BBa_K592100.
mTagBFP is based on the BioBrick BBa_K592100.
Lactose/IPTG inducible promoter with GFP reporter tagged with DAS-ssrA degradation tag followed by terminator. The tagged GFP is actively degraded within cell, thus provides better temporal resolution of green fluorescence and promoter activity. Presence of SspB is crucial for effective protein degradation.
pGAL1 + Flag + odr-10 + GFP + ADH1Te + pFUS1 + BFP + ADH1Te