Lorenzo Pasotti, Giulio Chiesa, Manuel Lupotto, Paolo Magni
tetracycline resistance protein TetA(C) (forward), [cf. BBa_J31006]
This part is the coding region of the TetR gene. It requires a promoter and RBS for the cell to be able to express tetracyline resistance.
TetA gene (DeOptimized from bp 1 to 141)
This sequence is the wild type TetA gene which has been deoptimized from base pairs 1 to 141. This is a basic part that can be placed behind a promoter and produce the Tetracycline resistance protein. Being that this gene is deoptimized, we are hypothesizing that it will allow for less translational efficiency as compared to the wild type TetA (no data is available at this time to support or reject this hypothesis).
During the design of this sequence, it was necessary to ensure that the deoptimized sequence did not contain BioBrick restriction sites and that the translational sequence was identical to that of the wild type gene.
This TetA gene has been deoptimized from base pairs 1 to 141 using the online Optimizer program (http://genomes.urv.es/OPTIMIZER/)and was constructed using customized Oligo segments that were annealed and ligated into a pSB1A2 vector.
P.chrysoporium lignin peroxidase isozyme H8 gene
Construct the measurement pathway of K1051208.
Contains a rather weak RBS in front of a codon modified variant of LacI. This reduces the likelihood of recombination with wild type LacI when used in multi plasmid systems.
The RBS was chosen to match the one in [Basu et al, 2005] in order to create a working band pass filter. Minimal RBS length was chosen to be 40 bp in accordance with [Salis et al, 2009].
Stop codon free EYFP in RFC[23] standard
Red fluorescent protein derived from DsRed. '''Advantages:''' * fast folding and maturation * bright and photo-stable
* gene synthesis * codon optimized for E. coli
Lipase ABC transporter recognition domain (LARD 1)
Jung Hoon Ahn from Korea Advanced Institute of Science and Technology
Lard 1 was designed for the secretion of fusion proteins.The LARD 1 included four glycine-rich repeats comprising a β-roll structure, and were added to the C-terminus of test proteins. Either Pro-Gly linker or Factor Xa site can be added between fusion proteins and LARD 1. Upon supplementation of E. coli with ABC transporter from E. chrysanthemi (ABC transporter PrtDEF), which function well in the phylogenetically-neighboring genus E.coli, EGF-LARDs were excreted into the culture supernatant.
In our designed LARD 1, we favoured not to add these cleavage site so if you want, yo can add either Pro-Gly linker or Factor Xa site. Secretion of fusion proteins with LARDs could be detected by Western blotting. Fusion proteins were traced in the culture supernatant using antibody against LARDs. Polyclonal antibodies against the C-terminal signal sequence were produced with a synthetic peptide (YQPTDRLVFQGADGST, residues 421???436 of TliA ,also of LARD).
LARD 1 composed of residues 303-476 of thermostable lipase (TliA) of Pseudomonas fluorescens.
This part is identical to BBa_I15010, the cph8 red light sensor. We were unable to transform BBa_I15010 from the the 2011 iGEM distribution kit. PCR attempts using VF2 and VR failed to amplify any DNA from the well sample. In the end, we chose to amplify cph8 from Jeffrey Tabor's pJT122 plasmid provided by Chris Voigt. The cph8 in pJT122 contained an internal PstI site, which was removed by point-mutagenesis. The resultant DNA sequence is identical to that of BBa_I15010.
The source DNA (pJT122) contained illegal restriction site (PstI) that had to be removed.
Biosynthesis/Oximes/CYP79B1/Plug 'n' Play
This BioBrick BBa_K527002 encode the plant enzyme Cytochrome P450 CYP79B1. Cytochromes p450 are defined as heme-thiolate proteins featuring a particular spectral signature at 450 nm, thus the name. Despite the label as cytochromes, these proteins are not involved in electron transfers, but act as monooxygenases in a wide range of reactions such as epoxidation, N-dealkylation, O-dealkylation, S-oxidation and hydroxylation. CYP79B1 is an N-hydroxylase converting L-Tryptophan into indole 3-acetaldoxime, the precursor of a cyanogenic glycoside . Oximes as the indole 3-acetaldoxime is generally toxic to fungi and is frequently used as a chemical fungicide. They function by inhibiting mitochondrial oxidases and thereby promote lipid peroxidation and the production of toxic reactive oxygen species. BBa_K527002 is submitted in the plasmid BBa_K678023 due to the different assembly method (Plug 'n' Play) hence the missing prefix and suffix.
Mutated at a Pst1 restiction site (nucleotide 342) No prefix and suffix. User assembly ends.
Institute of Plant Biology and Biotechnology Faculty of LIFE Science University of Copenhagen Denmark http://2011.igem.org/Team:DTU-Denmark-2
J23102 constitutive promoter + LovTAP Photosensor
LovTAP: Blue Light Photosensor and Transcriptional Regulator This photoreceptor was assembled by Strickland et al., and consists of a LOV (light-oxygen-voltage) domain of Avena sativa phototropin1 (AsLOV2) that senses blue light, fused to the trpR- DNA binding domain of the transcription factor trp repressor. The resulting protein is called LovTAP: LOV- and tryptophan-activated protein. LOV domains bind a flavin-mononucleotide (FMN) or flavin-adenine-dinucleotide (FAD) cofactor, which are used in a wide variety of metabolic pathways as cofactors in redox reactions and are available in most organisms. The cofactor has a broad absorption spectrum, with a maximum at 450 nm. Besides, the core of the LOV domain is often flanked by amino- or carboxy-terminal helices, termed A???α and Jα, respectively. In the LovTAP construction, AsLOV2 domain via its carboxyl-terminal Jα ???helix was ligated to an amino-terminal truncation of TrpR. The resulting protein has a domain-domain overlap with a shared helix. Thus, photoexcitation would change the conformation of the protein, in turn changing the stability of the shared-helix-domain contacts. Under the presence of light, absorption of a photon leads to the formation of a covalent adduct between the flavin mononucleotide (FMN) cofactor and a conserved cysteine residue in the AsLOV2 domain, which results in conformational rearrangements in LovTAP. This change impacts the affinity of the shared helix for the two domains: disrupting the contacts between the shared helix and the LOV domain and enabling the association of the shared helix with the TrpR domain, which establishes DNA-binding affinity and LovTAP can then bind DNA as an homodimer, repressing the transcription of the genes downstream of its binding sites. In the dark, when the shared helix contacts the LOV domain, the TrpR domain's DNA-binding affinity decreases and LovTAP is in an inactive conformation.
I-SceI homing endonuclease expression is controlled by pRha promoter. Expression is induced with L-rhamnose. I-SceI doesn't have an LVA degradation tag.
The system is leaky and meganuclease cut RS with high efficiency even without pRha induction.
Promoter pRha was amplified from the plasmid pJOE3075 (Dr. Altenbuchner). RBS was taken from an iGEM distribution plate (BBa_B0032). Meganuclease was amplified from the chromosome of SMR6316 E.Coli strain (Dr. Rosenberg).
Cyan fluorescent protein (CFP) in RFC25 standard
Tet Repressible C-terminal Arg Tagged EYFP (no terminator)
This is BBa_K331023 with the tetracycline repressible promoter BBa_R0040 added in front. This will be a component of a proof-of-concept part that will show localization of proteins into the interior of a microcompartment formed by the assembly of lumazine synthase proteins.
GoldenBraid destination vector alpha insert
E. coli flagellar protein linking torque machinery to cell wall; allows for induction of motility
A device to detected the level of negative supercoils in a circular plasmid.
E Coli promoter and iGEM kit plate CFP expression casette
activation domain B from SP1; Emamai et al. 1995
Du Jin,Hou Nan,Liu Duo,Yuan Fang,Zhang Weiran
coding the kanamycin resistance gene with a LVA tag,so the protein can degrade in a relative short time
link the sequence with the standard scar in iGEM
Lambda lysis cassette[BBa_K358004] with double terminator[BBa_B0015].
Red light sensor Cph8 (from Coliroid) with RBS
Chris French and Richard Edward Partridge-Hicks
Cph8 is a combination of cph1 and envZ which, together with PCB, form a transmembrane, histidine kinase-based light sensing system. It contains the photoreceptor from Cph1 and the histidine kinase and response-regulator from EnvZ???OmpR, producing a particularly strongresponse to light. Features: cph8 coding sequence 16 to 2250, RBS 6 to 10.
Notes: we were unable to revive BioBrick I15010 with the coding sequence of the cph8 red light sensor fusion protein, so we sought to amplify it from BioBrick BBa_M30109. This led to numerous problems. It is clear from the Registry sequence data that M30109 is not what it is supposed to be. It does include cph8 with a ribosome binding site, but the RBS is not joined to cph8 by a standard scar; promoter R0040 may be present, but there appears to be an EcoRI site a little upstream of the ribosome binding site so that, when we cloned our PCR product, we failed to recover the promoter region; also the PstI site in cph8, which was mutated out in I15010, is still present in the version in M30109. We therefore performed corrective PCR to obtain a clone with the correct prefix, a ribosome binding site, and the cph8 coding sequence with the PstI site properly removed, which we present here, cloned in pSB1C3.
BBa_I15010 (Photosensing Chimaera (Cph8) (not working, but amplified out of the whole construct))
This Cyan Fluorescent protein has been fused with 10 C-terminal Arginines to target it into the Lumazine-based microcompartment. Has a Ribosomal Binding Site
BioBrick CFP with Synthesized C-terminal Arg Tag
This chromoprotein, amilGFP, naturally exhibits very strong yellow color when expressed. The color is strong and readily visible to naked eye both in LB-culture and on agar plates. The DNA was provided by Jeffrey Miller at UCLA. It was made BioBrick-compatible after removal of two illegal internal restriction sites (EcoRI and PstI).
Two illegal internal restriction sites (EcoRI and PstI) was removed.
35s cauliflower mosaic virus + omega tobacco mosaic virus
Phycobilin synthesis operon without terminator
Phycobilin is chromophores (light-capturing molecules) found in cyanobacteria and in the chloroplasts of red algae, glaucophytes and some cryptomonads (though not in green algae and higher plants)， used in light sensor.
The actual forward sequence has the same changes at the start as seen in clone H11 described above. The reverse sequence is as expected. Features same as for the one above apart from the terminator.
This is derived from K098010 but PCR was used to remove the terminator, so that cph8 could be added at the end of the operon.
This is our protein domain Fok_a from our universal endonuclease, which we linked with an YFP-part.
Combiantion of our prtein domain Fok_a and the mVenus-YFP_FusionPartfrom the Team Freiburg07
Cerulean GFP is an optimized FRET donor molecule that was rationally designed by Piston and co-workers to eliminate the excited-state heterogeneity of ECFP. In ECFP, a single-exponential fit to the fluorescence lifetime is not feasible due to multiple components, whereas Cerulean exhibits essentially homogeneous excited-state decay kinetics, rendering this protein useful for lifetime imaging... Live cell imaging experiments have demonstrated that Cerulean GFP undergoes highly efficient FRET to yellow acceptor molecules (25, 26). Additionally, Cerulean exhibits more than twice the brightness of ECFP and CyPet, emitting light at fluorescence intensities similar to Citrine (20). ===References=== * Rizzo MA, Springer GH, Granada B, Piston DW. (2004) An improved cyan fluorescent protein variant useful for FRET. PMID: 14990965
* amino acid sequence taken from a pAG304GAL-Cerulean-ccdB from LindQuist lab * borders trimmed to match the construct used for PDB 2Q57 (last 9 AA are not resolved in PDB) * structure starts with MSK, this construct starts with MVSK but the M has been skipped for this Biobrick * gene synthesis * codon optimized for E. coli
mKate is a fluorophor derived from dsRed
mKate is a fluorophor derived from dsRed. It features the excitation maxima at 588 nm and the emission maxima at 633 nm.
Nucleotide sequence was added in order to avoid the formation of secondary structures during gBLOCK assembly and subsequent steps. The amino acid sequence was left unchanged.
IDT Technologies, gBLOCK assembly and subsequent PCR isolation
PoPS -> PhiR73 pag - PP promoter -> PoPS
This is a part that is composed of C-terminal end of chimeric flagellin (BBa_K133016). We used aminoacids from 215th aminoacid on. Chimeric flagellin is composed of C- and N-terminal part of E.coli flagellin and of variable region of H.pylori. The E.coli parts are responsible for binding and activation of cell membrane receptor TLR5 to initiate immune response, while H.pylori variable region was used to array the immune response against H.pylori. This part was used in combination with CF213 - chimeric flagellin (BBa_K133010). Between the CF213 and CF215 we have put H.pylori antigens in order to array the immune response against H.pylori.
It was important to split the chimeric flagellin in the variable region to assure that the antigen will be in the right place and not to interrupt the 3d structure of the protein.
We used polimerase chain reaction from chimeric flagellin (BBa_K133016).
ClyA is exported to the periplasm and exposed in outer-membrane vesicles
For fusion of proteins to the N-terminal extremity of ClyA using the silver fusion standard RFC23
promoter of phoB in E.coli, an external phosphate regulated promoter which can be activated when encountering phosphate limitation.
The promoter region should contain PhoB binding site
putative type III polyketide synthase (PKS) gene
Aptamer-CheZ: theophylline-sensitive synthetic riboswitch+phosphatase which dephosphorylates CheY-P
This part is composed of a theophylline-sensitive aptamer,which will pair the ribosome binding site of the downstream gene in the absence of theophylline and thus inhibit the translation of the gene while expose the ribosome binding site in the presence of theophylline, and the CheZ gene, which is a phosphatase which dephosphorylates CheY-P and causes the flagellum to rotate counterclockwise and is essential for keeping the motility of E.coli. Thus this part provides a ligand-inducible motility and makes it possible to guide E.coli by theophylline.
We found the sequence of this part in literature and tried to design primers to construct it. But we were faced with a challenging problem in primer design, which comes from the basic properties of the aptamer involved. The aptamer tends to pair the ribosome binding site in the absence of theophylline, which is true in PCR, and enables primers to pair to the corresponding parts and the new strand to elongate. We made contact with the author of our reference and found they took different strategies in assembling the aptamer to CheZ instead of PCR. But thanks to the warm help of Professor J. S. Parkinson and Professor J. P. Gallivan, we acquired the sequence of primers used in assembling the aptamer to another gene and designed our own after analyzing them: forward primer:5'-GTTTCGAATTCGCGGCCGCTTCTAGGGTGATACCAGCATCGTCTTGATGCCCTTGGCAGCACCCCGCTGCAAGACAACAAG ATGCAACCATCAATCAAACC-3' reverse primer:5'-GTTTCCTGCAGCGGCCGCTACTAGTATTATTAAAATCCAAGACTATCCAACAAATCGT-3' Besides, we used special protocal of PCR: 96℃ 10min 96℃ 30s, 56℃ 30s, 72℃ 1min for 16 cycles and each cycle the annealing temperature increases by 1℃ 96℃ 30s, 72℃ 30s, 72℃ 1min for 20 cycles 72℃ 10min
Escherichia coli, the strain has always been kept in the lab of USTC-IGEM
Mutated from BBa_C0051 Repressor, Lambda cI (RBS- LVA+) to be temperature sensitive and UV insensitive.
First attempt to mutate failed. Suspect the problem is a low mutation rate due to results of positive control. May be a result of the mutagenesis kit being old. Second attempt to mutate succeeded. However, the sequencing had a lot of bad sequence at the beginings, so that the ind- mutation was not sequenced by either of the primers used. The ind- mutation was verified by the presence of a HindIII restrition site. This part was never completely sequenced. The cI857 QPI was made by Blue Heron, and this was part was abandoned.
Hendrix, Roger W. et al., ed. Lambda II. New York: Cold Spring Harbor Laboratory, 1983. ind- mutation on p. 626 (at bp 37589 of Lambda) 857 mutation on p. 628 (at bp 37742 of Lambda) Made by introducing two point mutations to Lambda cI (BBa_C0051): G(352)->A for ind- and G(199)->A for 857 temperature sensitivity.
amilGFP(+LVA) generator(+RBS, +terminator)
Lumazine Synthase with Transciptional Terminator (No Promoter)
Composite part: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K249002 BBa_K249002] + [http://partsregistry.org/wiki/index.php/Part:BBa_B0010 BBa_B0010] + [http://partsregistry.org/wiki/index.php/Part:BBa_B0010 BBa_B0010] + [http://partsregistry.org/wiki/index.php/Part:BBa_B0012 BBa_B0012] Made by assembling [http://partsregistry.org/wiki/index.php?title=Part:BBa_K249002 BBa_K249002] with [http://partsregistry.org/Part:BBa_B0015 BBa_B0015] Since this part is lacking the promoter, Lumazine Synthase production may be regulated by the addition different promoters upstream. Regulation of Lumazine Synthase will be dependent on the promoter being utilized.
Intermediate part to assemble the "Co-localization Construct".
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K249002 BBa_K249002] and [http://partsregistry.org/Part:BBa_B0015 BBa_B0015] were obtained from iGEM distribution plates.
I-SceI homing endonuclease expression is controlled by pBad promoter. Expression is induced with IPTG if LacI is present in the cell. I-SceI doesn't have an LVA degradation tag.
The system is leaky and meganuclease cut RS with high efficiency even without pBad induction.
Promoter pBad and RBS was taken from an iGEM distribution plate (BBa_I13453 and BBa_B0032). Meganuclease was amplified from the chromosome of SMR6316 E.Coli strain (Dr. Rosenberg).
constitute CMV promoter;PCR cloned from pcDNA3.1+
Syed Habib Tahir Bukhari, Ashwini Rahul Akkineni, Adithya Ananth, Victor Gordeev, Svea Grieb, Sarah Mansour, Mareike Roth, Charanya Sampathkumar, Lucas Schirmer, Jonathan Tam
This part combines a Tetracycline repressive promoter, RBS and the reporter ecfp (cyan fluorescent protein).
Will express ecfp constitutively without repression of pTet
This chromoprotein eforRed naturally exhibits red color when expressed. The color is weaker than RFP, however. On agar plates and in liquid culture, the color is readily visible to naked eye in less than 24 hours of incubation. The DNA was codon-optimized for expression in E.coli and synthesized by the Korean company Bioneer Corporation.
Construct the measurement pathway of K1051207.
Coding region for Prepro-Thaumatin. Codon optimized for yeast.
Synthesized by IDT. The part is codon optimized for yeast. 3 forbidden restriction sites have been removed.
Based on: http://www.ncbi.nlm.nih.gov/nuccore/121945717
A composite part between J23100 and sfGFP
[R0010][B0034] - RFP-FliC Insertion Chimera
This is the modified RFP protein obtained from J04450, that has been incorporated into the E. coli flagellin as an insertion. This part can be used as a template for making other PCR overlap insertions. After transforming the product, and digesting the template plasmid with Dpn1, any template that has not been successfully digested will form red colonies.
The linker and D3 overlap sequences are standard for our parts. By digesting the plasmid with the two inner enzymes, SpeI and NheI for our BB-2 standard parts, you can purify and then PCR overlap extend using the following protocol to result in a brand new chimera.
This was created using overlap extension PCR to insert into K781000 at the annotated site. The insert sequence was amplified using PCR from J04450.
mCherry is a red fluorescent protein used for analysis of localisation of protein.
Chloramphenicol resistance protein - Terminator
Chloraphemicol acetyltransferase protein( BBa_J31005 ) coupled to the double terminator ( BBa_B0015 ). Chloraphemicol acetyltransferase confers resistance to Chloraphemicol. The double terminator is the most commonly used terminator and is a combination of parts BBa_B0010 and BBa_B0012 . The double terminator allows the CAT to be incorporated into a closed transcriptional unit.
Chloraphemicol acetyltransferase is an exisiting registry protein. The double terminator is the most commonly used registry termiantor.
The Chloraphemicol acetyltransferase and double terminator were taken both taken from the registry.
Antimicrobial peptide generator (LL-37 with codon usage for E. coli)
Specific codon usage for E. coli. It works under arabinose induction.
The 2 component system that responds to ethanol that activates transcription of pexaA.
Each part contains a RBS but the part does not contain a promoter.
Membrane protein involved in type I secretion. When protein is fused to the HlyA signal peptide, this protein aids in protein secretion.
Codon optimized for expression in E. coli while avoiding BioBricks standard restriction sites.
lamB is the a porin protein which is part of the maltose pathway. It is also the receptor associated and required fro lambda phage infection.
Alkaline phosphatase coding sequence (no rbs, promoter, or terminator present) from Citrobacter. This bacterium's phoA was chosen due simply to the absense of Biobrick restriction sites.
pre-pcr ca1033F/ca1032R on Citrobacter gen (1500 bp) PCR ca1032F/R on above PCR (1500 bp, XbaI/EcoRI) Sub into pSB1A2-Bca1008 (XbaI/MfeI, 2077+935, large) Product is pSB1A2-Bca1032 ---- ca1032F Forward oligo to biobrick phoA of Citrobacter GGTTAtctagaTGTTTTTTATTTTAACTGTATTTATACATGGAG ca1032R Reverse oligo to biobrick phoA of Citrobacter cagtaGAATTCccagaggcggcaTTACTTCAG ca1033F Forward constant oligo to clone phoA ttatcatcgctccgcatcctg
Hybrid promoter (trp & lac regulated -- tac pR)
The Ptac promoter is a functional hybrid promoter, derived from the trp and lac promoters, that are regulated by trp and lac [1]. The Ptac sequence has been extracted by PCR from the commercially available plasmid pMAL-pIII from New England Biolabs (N8101S), with the addition of the biobrick prefix and suffix sequences. [1] Proc. Natl. Acad. Sci. USA, Vol. 80, pp. 21-25
Sequence includes lacIq and Shine-Dalgarno sequence from pMAL-pIII prior to the Ptac promoter to ensure correct functionality of the promoter.
Heme Oxygenase with T7 & RBS (E. coli Codon Optimised)
E. coli codon optimised Synechocystis Heme Oxygenase with a Ribosome Binding Site (RBS) and T7 promoter ( Part BBa1719005 , oxidises Heme into biliverdin which absorbs light in the 360 nm and 650 nm wavelength producing a green colouration.
GC rich regions, which formed hairpin loops in synthesis, were removed. Restriction sites were removed to conform to biobrick standards.
The part was codon optimsed (http://www.bioinformatics.org/sms2/rev_trans.html) from Synechocystis and assembled using Gibson Assembly. Select GC rich regions were removed.
lambda lysozyme with native rbs site in BBb format. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0006/al0002 on pSB3C6-Bjh1501 (527bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112305 ----------------------------------------------- al0006 Forward Biobricking of lambda lysozyme with rbs site CCATAGAATTCatgAGATCTaaaaaagccggagtagaagatgg al0002 Reverse Biobricking of lambda lysozyme with rbs site CGttaGGATCCtcatacatcaatctctctgaccgttccgcc
Mutated flagella master regulator (FlhDC mut).
The master regulator operon for flagellum synthesis. Produces transcriptional factors for Class II flagellar regulon genes.
The sequence comes directly from E.Coli.
Changed a T to C to remove Pst1 restriction site.
Secretion Signal and Streptavidin Binding Tags
This part is the vector into which afp is inserted to tag it for purification using the IPP system. It contains an NheI restriction site that allows for the insertion of the desired protein coding sequence (NheI is compatible with XbaI and SpeI). This Target Vector part contains two tags: a secretion tag that causes the protein to be exported to the extracellular space, and a nanotag that causes the protein to bind to stretavidin (or some analog). For this part to function properly, it must be combined with a promoter and RBS, and used in E.coli strain UW5alpha, which has two other plasmids containing the other genes required for proper secretion and display of the protein to be purified on the outer membrane.
NheI was chosen as the protein insertion site because it is compatible with digested XbaI and SpeI restriction sites. The TEV sites and HIS tags flanking the NheI site are present to separate the inserted protein from the added tags. GSA linkers were used to separate the various features of the part.
Nanotag (bases 1-48): From Lamla and Erdmann (http://www.ncbi.nlm.nih.gov/pubmed/14680960). Secretion tag (last 181 AA): Gene Bank ID Erwinia chrysanthemi M60395.1
This part encodes a Δ6 desaturase derived from Synechocystis sp PCC 6803. The enzyme is a membrane-bound protein able to introduce a double bond at the Δ-12 site in the hydrocarbon chain of oleic acid (18:1, Δ9). This converts the substrate into linoleic acid (18:2 Δ9,12), a polyunsaturated fatty acid (PUFA). Mass spectrometry results show that feeding oleic acid to transformed E. coli, the bacterial membrane composition changes. Linoleic acid is observed.
This desaturase comes from Synechocystis sp PCC 6803 genome.
DNA-invertase Hin from Salmonella typhimurium
Generates the Salmonella typhimurium Hin protein. This protein is used to invert DNA that is flanked by Hix siteds.
arabinose regulator (araC) coding sequence
This sequence is a copy of the sequence 70,387 -> 71,265 in E. coli K-12 MG1655 which is the coding sequence of the pBAD repressor araC. From ECOCYC: The "arabinose regulator," AraC, is a transcription factor that regulates transcription of several genes and operons involved in arabinose catabolism and transport. It coregulates with another transcriptional regulator, CRP; both are transcription factors involved in l-arabinose degradation. These regulators bind cooperatively to activate transcription of five operons related to transport, catabolism, and autoregulation of l-arabinose. Transcription of these operons is induced when E. coli is grown in the absence of glucose and when the physiological inducer, l-arabinose, binds to the AraC regulator. In the absence of glucose, cellular cyclic AMP levels are high and cyclic AMP forms a dimeric complex with CRP to coregulate with AraC. For more info go to http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10054
The part was amplified from chromosomal DNA with primers carrying prefix and suffix overhangs
This chromoprotein from the coral Galaxea fascicularis, gfasPurple, naturally exhibits strong color when expressed.
Synthesized and codon optimized for E coli by GenScript.
Encodes a peptidase from proteinase K family, breaks down polylactic acid
This is the mCherry fluorescent protein, yeast-codon optimized and biobricked in the silver lab BioFusion standard for use in fusion proteins.
LacI transcriptional repressor protein ( BBa_K143033 ) coupled to the double terminator ( BBa_B0015 . The LacI does not possess a LVA degradation tag and gas a short (3 amino acid) N-terminal deletion consistent with LacI used in conjunction with ''B. subtilis''. LacI can be used in conjunction with the lac operon promoter ( BBa_K143015 ), where the LacI will act as a receiver for an IPTG input to result in a '''Polymerases per second''' (PoPS) output. The double terminator is the most commonly used terminator and is a combination of parts BBa_B0010 and BBa_B0012 . The double terminator allows the LacI to be easily incorporated into a closed transcriptional unit.
LacI was identified from the pDR111 ''B. subtilis'' integration vector. The double terminator is the most commonly used registry terminator.
LacI was produced by PCR cloning using Pfu form the ''B. subtilis'' integration vector and cloned into a BioBrick with the registry double terminator.
TEV protease S219P autocatalysis resistant variant
TEV protease S219P autocatalysis resistant variant Introduction : This is the nuclear inclusion protease, endogenous to Tobacco Etch Virus and is used in the late lifecycle to cleave polyprotein precursors. The recognition sequence is ENLYFQG/S [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-45PMGK3-9P&_user=217827&_coverDate=02%2F01%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=217827&md5=e075aad3a349720ad9484095d01a65be&searchtype=a]] between QG or QSDue to it???s stringent sequence specificity, TEV is commonly used to cleave genetically engineered proteins. Uses: TEV proteinase is used to cleave fusion proteins. It is useful due to its high degree of specificity http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-45PMGK3-9P&_user=217827&_coverDate=02%2F01%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=217827&md5=e075aad3a349720ad9484095d01a65be&searchtype=a and potential to be used in vivo or in vitro applications. Auto-inactivation Wild type TEV protease also cleaves itself at Met 218 and Ser 219 http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WXR-45R86R6-5X&_user=7635175&_coverDate=06%2F20%2F1995&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=7635175&md5=0a1521e783c0bd4126b145f3f6d766d4&searchtype=a . This leads to auto-inactivation of the TEV protease and progressive loss of activity of the protein. The rate of inactivation is proportional to the concentration of protease http://peds.oxfordjournals.org/content/14/12/993.long More stable Mutants have been produced by single amino acid substitutions S219V (AGC(serine) to GTG(valine) and S219P (AGC(serine) to CCG(proline) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency '''Table I.''' Kinetic parameters for wild-type and mutant TEV proteases with the peptide substrate TENLYFQSGTRR-NH2. Enzyme Km (mM) kcat (s−1) kcat/Km (mM−1 s−1) Wild-type 0.061 ?? 0.010 0.16 ?? 0.01 2.62 ?? 0.46 S219V* 0.041 ?? 0.010 0.19 ?? 0.01 4.63 ?? 1.16 S219P* 0.066 ?? 0.008 0.09 ?? 0.01 1.36 ?? 0.22 S219P* - virtually imperivious to autocatalysis S219V* - retains same activity as wild type Full article can be seen here http://peds.oxfordjournals.org/content/14/12/993.long
The part was produced by nucleotide synthesis by mwg ??? eurofin
TEV protease is naturally found in Tobacco Etch Virus genome. This part contains the sequence codon optimized for expression in B.subtilis using mwg ??? eurofins www.eurofinsdna.com proprietary software.
This Cyan Fluorescent protein has been fused with 10 N-terminal Arginines to target it into the Lumazine-based microcompartment. Has a double terminator
BioBrick CFP with synthesized N-terminal Arg Tag
GoldenBraid destination vector omega insert
LacI repressor (temperature-sensitive mut 265) (+LVA)
Mutated from BBa_C0012 Repressor, LacI (LVA+) to be temperature sensitive. Mutation from Chao, et al., 2002: Gly265->Asp (GG(803)T->GAT)
37℃ induced RBS + tetR + double terminator
It is a transcriptional unit including 37℃ induced RBS + tetR + double terminator. It can be added various promoter to adapt your conditions.
To regulate following genes by temperature. To maintain cells at 37?? C ↑ : the tetR protein be generated and repress Ptet's circuit. To release cells at 37?? C ↓ : the tetR protein not be generated and do not repress Ptet's circuit.
the 37℃ induced RBS cames from iGEM08_TUDelft
This part codes for the fusion of Pyruvate decarboxylase and Alcohol dehydrogenase from ''Zymomonas mobilis''. Fused enzymes increase ethanol yields and enable its faster production.
The part is based on the BBa_K173003 - the enzymatic components of this BioBrick were fused using MABEL technology. The fusion removed start codon in adhB, stop codon in pdc and the RBS inbetween.
''Z. mobilis'' gDNA codon optimised for ''E. coli''
AraC protein(reversed sequence) and Pbad promoter
This part contains AraC protein generator(BBa_K228008) and Pbad promoter(BBa_I13453). The expression of any coding sequence following the Pbad promoter is repressed constitutively by AraC protein and induced by L-arabinose.
The maximum concentration of L-arabinose we tested is 10^-4 M.
RBS + SlrP secretion tag, salmonella T3 sec. sys.
leucine-rich repeat protein (SlrP) One of the secretion signals in Salmonella Type 3 Secretion System(T3SS). In theory can be used to secrete tagged proteins through Type 3 Secretion Apparatus(T3SA). Due to functional restrictions of T3SA it's known that it can't transport very stable proteins like zinc fingers. Regardless it was shown to transport such stable proteins like GFP. Currently this part is in trials to see if it will secrete GFP. Chassis is E.coli with BAC vector fragment (SGSC4024 1464540-1562427) of salmonella SPI2. Please consult papers bellow about details A conserved amino acid sequence directing intracellular type III secretion by Salmonella typhimurium Edward A. Miao and Samuel I. Miller 2000
RBS was added by primer in the process of PCR.
This parts source is from genomic sequence of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2. Amplified b y PCR.
Synthetic periplasmic binding protein that docks a vanillin molecule
lacI:LVA device (constitutive promoter, RBS and terminator)
The trancsriptional repressor lacI with LVA tag for faster degradation (BBa_C0012)under the constitutivly active promoter (BBa_J23106), with a RBS (BBa_B0030) and with a terminator (BBa_B1002). This device can be used to overexpress lacI with the purpose of repressing transcription from high-copy plasmids containting the lac promoter (BBa_R0010). The repression can be relieved by allosteric binding of IPTG to lacI, thereby promoting transcription from the lac promoter.
Used a strong terminator so that the composite part can be used in front of the lac promoter without unwanted transcription of the regulated gene.
The source of the part will be its components.
Construct the measurement pathway of K1051206.
Dihydrodipicolinate synthase is the first enzyme unique to lysine biosynthesis, catalyzing the condensation of pyruvate and aspartate β-semialdehyde by a ping-pong mechanism. It also catalyzes the rate-limiting step in E.coli lysine biosynthesis after aspartate kinase III. The condensing enzyme-pyruvate complex, prior to reduction and hydrolysis, exists as the Schiff base formed between the carbonyl of pyruvate and an ε-amino group of a lysine residue on the enzyme. This property classifies the enzyme as a Class I aldolase with respect to carbon-carbon bond formation. The enzyme is inhibited by lysine, a feedback inhibitor. (source: Ecocyc)
Engineered pH-inducible intein (codon optimized for E. coli) - internal domain
Codon optimized for E. coli. The amino acid sequence comes from [1].
The original sequence comes from M. tuberculosis genomic DNA (RecA gene), but it has been highly engineered (according to [1]) to create a pH-inducible protein domain.
This sequence codes for the LsrR gene which is found in the E. coli strain MG1655. When this protein fors a complex with phosphorylated AI-2 it promotes the LsrA promoter.
The DNA for this part is found in the E. coli strain MG1655.
J23105 constitutive promoter + LovTAP Photosensor
This photoreceptor was assembled by Strickland et al., and consists of a LOV (light-oxygen-voltage) domain of Avena sativa phototropin1 (AsLOV2) that senses blue light, fused to the trpR- DNA binding domain of the transcription factor trp repressor. The resulting protein is called LovTAP: LOV- and tryptophan-activated protein. LOV domains bind a flavin-mononucleotide (FMN) or flavin-adenine-dinucleotide (FAD) cofactor, which are used in a wide variety of metabolic pathways as cofactors in redox reactions and are available in most organisms. The cofactor has a broad absorption spectrum, with a maximum at 450 nm. Besides, the core of the LOV domain is often flanked by amino- or carboxy-terminal helices, termed A???α and Jα, respectively. In the LovTAP construction, AsLOV2 domain via its carboxyl-terminal Jα ???helix was ligated to an amino-terminal truncation of TrpR. The resulting protein has a domain-domain overlap with a shared helix. Thus, photoexcitation would change the conformation of the protein, in turn changing the stability of the shared-helix-domain contacts. Under the presence of light, absorption of a photon leads to the formation of a covalent adduct between the flavin mononucleotide (FMN) cofactor and a conserved cysteine residue in the AsLOV2 domain, which results in conformational rearrangements in LovTAP. This change impacts the affinity of the shared helix for the two domains: disrupting the contacts between the shared helix and the LOV domain and enabling the association of the shared helix with the TrpR domain, which establishes DNA-binding affinity and LovTAP can then bind DNA as an homodimer, repressing the transcription of the genes downstream of its binding sites. In the dark, when the shared helix contacts the LOV domain, the TrpR domain's DNA-binding affinity decreases and LovTAP is in an inactive conformation.
Aldehyde dehydrogenase from Geobacillus thermoleovorans B23.
Nucleotide sequence optimized for expression in E.coli.
HRT2 prenyltransferase from Hevea Brasilianis (ara promoter without araC: arabinose inducible)
This part encodes the rubberproducing prenyltransferase from the rubber tree Hevea brasiliensis. It uses isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) as substrates to produce cis-1,4 polyisoprene natural rubber. The gene is expressed from the arabinose promoter (BBa_I13453), which is induceable with arabinose under glucose scarce conditions. Note that the regulater insn't a part of this part and thus no/very little transcription is expected.
There is a 3xFLAG-tag after the HRT2 stop codon, and thus the 3xFLAG-tag isn't translated.
this is the intermediate construct combining pBAD with BBa_S0100, so as to express lacI gene
This part was synthesized from IDT and is native to Staphylococcus aureus
Part to produce C. braakii phytase. This phytase acts to break down phytic acid into inositol, releasing chelated iron and other micronutrients.
http://www.ncbi.nlm.nih.gov/nuccore/AY471611.1 Genomic sequence
Lorenzo Pasotti, Susanna Zucca, Paolo Magni
SST (BBa_K899001) with double RBS (BBa_K899010)
J23114 constitutive promoter + LovTAP Photosensor
Expresses phasin fused to the HlyA signal peptide. Secretion target.
this part is chimeric protein of the enzyme relating propandiol metabolize and green fluorescent protein. this part was localize to BMC.
This part contains the cheZ operon coding region without an rbs nor a terminator. cheZ protein is responsible for the dephosphorylation of cheY protein in bacteria flagella movement.
This part is without promoter, rbs or terminator.
The origin of the cheZ gene comes from E. Coli strain MG1655 chromosome.
It generates AraC regulatory protein which binds to and repress the pBad promoter(such as BBa_I13453). L-arabinose, which acts as inducer, could efficiently prevent the protein binding to pBad.
This is the pyruvate oxidase from Streptococcus pneumoniae R6. It catalyzes the formation of hydrogen peroxide by oxidizing pyruvate in the reaction: pyruvate + Pi + O2 --> acetyl-P + CO2 + H2O2.
Removal of a PstI site very near the 5' end with PCR primers.
Genomic PCR from a prepared genome of Streptococcus pneumoniae R6 from ATCC (# BAA-255D-5)
p(LacI) controlled I-SceI homing endonuclease generator
p(LacI) controlled I-SceI homing endonuclease generator, inducible with IPTG when LacI is present in the cell
BBa_K142205 is wrong, so this part was assembled again.
Assembled with BBa_K142202 from iGEM08_ETH_Zurich.
BBa_E0032 EYFP (RBS-, LVA+) and B0015 (Intermidiate Building Sequence)
This is an EYFP coding sequence plus a LVA tag and the Terminator Sequence B0015
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.
ADH, an alcohol dehydrogenase isolated from Bacillus thermoleovorans B23, is capable of converting n-alkanols into the corresponding n-alkanal, the second step in the biodegradation of alkanes.
Stop codon free ECFP in RFC[23] standard
The Targeted Biobrick is a specially constructed for cell-specific attachment and subsequent internalization. It consists of a TAT peptide for attachment (can be modified by other peptide for different specificity), the selected segment in fiber shaft of adenovirus and RGD sequence. Different modules or domains are linked by linker sequences.
This sequence is the wild type TetA gene which has been optimized from base pairs 1 to 141. This is a basic part that can be placed behind a promoter and produce the Tetracycline resistance protein. Being that this gene is optimized, we are hypothesizing that it will allow for greater translational efficiency as compared to the wild type TetA (no data is available at this time to support or reject this hypothesis).
During the design of this sequence, it was necessary to ensure that the optimized sequence did not contain BioBrick restriction sites and that the translational sequence was identical to that of the wild type gene.
This TetA gene has been optimized from base pairs 1 to 141 using the online Optimizer program (http://genomes.urv.es/OPTIMIZER/)and was constructed using customized Oligo segments that were annealed and ligated into a pSB1A2 vector.
K1051001 (non stop codon ECFP + K1051006 (Stop codon + TBY-1 terminator)
Yeast terminator TBY-1 joint with red flourescent protein. After added with 23 suffix and preffix, this composite biobricks can be joint with other coding gene sequence in our project. Thus it would be a reporter showing red flourescent,also as a terminator which can stop the transcription.
Saccharomyces cerevisiae genomics sequence
a constitutive expression promoter or use in mammalian cells. Ribosome binding site is included.
First attempt to mutate failed. Suspect the problem is a low mutation rate due to results of positive control. May be a result of the mutagenesis kit being old. Second attempt to mutate failed. Sent out to Blue Heron for synthesis.
Beta-lactamase with His Tag under the control of the inducible lacI promoter
We used a linker attached to a protein membrane "estA"
The mRFP coding sequence in the reverse orientation. This part requires a reverse RBS and reverse promoter to be expressed
RFP reverse was PCR amplified from mRFP (BBaE1010) using the following primers: 5'-atgcactagtatggcttcctccgaagacgt "Spe mRFP F" 5'-gcattctagattaagcaccggtggagtgac "Xba mRFP R"
RFP reverse was PCR amplified from mRFP (BBaE1010).
The part contains RFP sequence from E1010 with removal of Kpn I site and stop ending changed to tgatga
Green fluorescent protein (GFP) is a 27 kDa protein of 238 amino acids in length. GFP is intrinsically fluorescent and hence is ideal as a non-invasive marker in living cells. It is used as a cell lineage tracer, reporter of gene expression, a measure of protein-protein interactions, and as an intracellular pH indicator. GFP can function as a protein tag as it tolerates N- and C-terminal fusion to a broad variety of proteins (Chin et al.). Wild-type GFP fluoresces maximally when excited at 395 nm, with a minor peak at 475 nm. Fluorescence emission peaks at 509 nm. It is stable within a range of pH 5.5 to 12. Denaturation occurs at temperatures above 70??C (Chin et al.). Several variants with improved absorption or emission spectra, or increased the fluorescence intensity, have since been developed. Variants also exhibit different thermosensitivities (Chin et al.). As stated before, the maximum excitation spectra of GFPuv is 395nm while its maximum emission spectra is 509nm, which is similar to that of wild-type GFP. Compared to wild-type GFP, GFPuv contains three amino acid substitutions (Phe-99 to Ser, Met-153 to Thr, and Val-163 to Ala [based on the amino acid numbering of wild-type GFP]), none of which alter the chromophore sequence. The main advantage of this variant over wild-type GFP is its large amount of fluorescence when excited by ultra-violet (UV) light at 395nm. GFPuv is 18 times brighter than wild-type GFP under ultra-violet excitation and can easily be detected by the eye without the need for specialized equipment. Furthermore, GFPuv expressed in E. coli is a soluble, fluorescent protein in contrast to wild-type GFP, much of which may be expressed as a nonfluorescent protein in inclusion bodies. Consequently, the GFPuv gene is expressed very efficiently in E. coli (Chin et al.). Reference: Chin et al. Characterization of Green Fluorescent Protein. Retrieved from: http://staff.science.nus.edu.sg/~scilooe/srp_2003/sci_paper/dbs/research%20_paper/ngeow_kao_chin.pdf
The sequence complied with RFC-10 assembly compatibility as a requirement for Utah State University Synthetic Biology class, so no design considerations had to be taken into account during the creation of the sequence as a biobrick.
GFPuv comes was originally isolated from the bioluminescent jellyfish Aequorea Victoria (Chin et al.).
The promoter is activated when: (1) there is no glucose in the medium and (2) there is lactose in the medium. Our construction uses the well-known lactose operon system. A lack of glucose promotes the formation of CAP (or CRP), which binds to specific sites upstream of sugar-metabolization genes and activates its transcription. Moreover, another condition is needed since LacI repressor will bind to the operator region and will block the transcription unless lactose is also in the medium.
The sequence of the promoter comes from E. coli genome, whereas the sequence encoding the AmCyan fluorescent protein is not natural, since it was optimized for codon usage and maximum fluorescence production.
CCMV wt coat protein under IPTG induced promoter
this part is created with a part from the parts registry, and a part made by Wageningen_UR 2012 with the template of Dr. Kormelink
This part is a delta-12 desaturase derived from Synechocystis sp PCC 6803. The enzyme is able to introduce a double bond at the Δ-12 site in the hydrocarbon chain of oleic acid (18:1 , Δ9), for its conversion into linoleic acid (18:2 Δ9,12), a polyunsaturated fatty acid (PUFA). Mass spectrometry results show that when feeding oleic acid to E. coli transformed with this part, the bacterial membrane composition is changed, and linoleic acid is observed.
Medium constitutive expression of EBF synthase from Artemisia annua
For the production of the alarmhormone,(E)-β-farnesene, a sesquiterpene synthase gene (GenBank Accession No. AJ271793) was put behind a medium (constitutive) promoter and medium RBS (BBa_K608006).
no EcoRI, XbaI, NotI, SpeI or PstI sites present
Composite part; genomic sequence from EBF synthase from Artemisia annua combined with iGEM bricks. BBa_K608006 + Bba_K1060002 + Bba_B0015
Bas Stringer (Sequence by Paul van Dieken)
* The ''fliC'' gene codes for flagellin. This protein is the subunit that forms the flagellar filament of ''E. Coli''.
* The ''fliC'' gene contained three PstI sites and one SpeI site that had to be removed. These mutations were induced with different primers via overlap-PCR. * ''flhDC'' was amplified from genomic DNA of E. coli DH10B via PCR using the following primers: ** Fwd: ACTGAATTCGCGGCCGCTTCTAGATGCATACCTCCGAGTTGCTG ** Rev: TCCTGCAGCGGCCGCTACTAGTTACTGCCCGGGATGGCGGTTGACATAAGCcGCAGGCAAAGCTGCCAACAG
* The part was amplified from genomic DNA of E. coli str. K-12 substr. DH10B, complete genome (CP000948.1).
Lac-inducible generator of Lpp-OmpA(46-159)-Streptavidin wild-type + His6 tag
This part codes for the core part of wild-type streptavidin protein, without the start codon. Streptavidin protein binds strongly to the biotin molecule, and can be used to bind biotinylated nucleic acids or peptides. There is a histidine tag added at the end of the coding sequence right before the stop codons. You can probe for this protein with an anti-his(C-term) antibody. It was used as part of a fusion protein with lipoprotein signal peptide and outer membrane protein A transmembrane domains, to express streptavidin on the outer surface of E. coli.
There was originally a XbaI site TCTAGA at nucleotides 118-123 of the part, which was mutated to AGCCGC. It is missing its start codon at the beginning, as the intention was to fuse this with surface display domains upstream. There are no spacer nucleotides between the part and the XbaI restriction site, or between the part and the SpeI restriction site, so that the mixed site would be 6bp long, and reading frame would be maintained between protein domains.
PCR off a plasmid obtained from Alice Ting's lab at MIT.
phaB BioPlastic polyhydroxybutyrate synthesis pathway (origin PCC6803 slr1993)
This neatly designed color coding cassette is a prime candidate to replace BBa_J04450, the current RFP cassette in all of the BioBrick backbones. Bacteria colonies exhibit strong blue color after 18 hours. Due to the much darker blue/purple color tone, even partially blue colonies are readily recognized. Promoter is constitutive, the RBS is strong, the double terminator is not B0015 but B0014.
B0014 instead of B0015 in order to avoid unwanted primer binding, etc. J23101 promoter to ensure strong and constitutive expression at all times.
amilCP comes from the genome of Acropora millepora. All other parts are assembled from the iGEM 2011 distribution kit.
resister this part since the sequence of BBa_K082026 is wrong.lacIM1 is a codon-modified lacI designed to reduce the likelihood of recombination with the wild-type lacI(Subhayu Basu,et al.,2005).
This Yellow Fluorescent protein has been fused with 10 N-terminal Arginines to target it into the Lumazine-based microcompartment. Has a double terminator at the end
BioBrick YFP with synthesized N-termianl Arg Tag
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
Sequence for pTet inverting regulator driven by the TetR protein.
BBa_R0040 TetR-Regulated Promoter is based on a cI promoter. It has been modified to include two TetR binding sites and the BioBrick standard assembly head and tail restriction sites.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210.
This is a sensitivity tuner created form the C protein and Pmom promoter of the Mu bacteriophage it can be used to increase the PoPS of a construct. The C protein encoded in the first part of the construct is the activator of the Pmom promoter, which does not begin transcription until the C protein binds to its consensus sequence and attracts the polymerase.
We built this part form C and Pmom sequences of Mu bacetriophage and included the bidirectional terminator BBa_B0014 from the registry.
Protein domain (active) of the restriction endonuclease FokI
This part is used as the active domain of our universal restriction endonuclease. It cut double stranded DNA, when it fused with the inactive protein domain of our universal restriction endonuclease(BBa_K243001).
Modifications of the vector (catalytic active heterodimer) -heterodimeric aminio acids * switch Glutamate 490/310-312 to Lysin (GAA->AAA) * switch isoleucin 538/454-456 to Lysin (ATC->AAA)
extract coding region of Fok from the restriction-modification genes of the chromosomal DNA of Flavobacterium okeanokoites. Part synthesized by Mr.Gene
Red fluorescent protein with assembly standard 23 is engineered mutant of red fluorescent protein from Discosoma striata (coral).
You have to remove the termination codon before you use it
engineered mutant of red fluorescent protein from Discosoma striata
Giacomo Zambianchi, Alessandro Ranieri, Manuel Lupotto, Paolo Magni
It is identical to BBa_K208001 , but it lacks the stop codon in order to support protein fusions. It has been designed as a head domain: The Prefix sequence is 5'-GAATTCGCGGCCGCTTCTAG-3' (RFC10 Prefix) The Suffix sequence is 5'-ACTAGTAGCGGCCGCTGCAG-3' (RFC23 Suffix) For these reasons, a tail domain or an internal domain (compatible with RFC23) are ready to be assembled downstream to create protein fusions. CONSTRUCTION METHOD: BBa_K208001 (provided by iGEM HQ in pSB3K3 in RFC23 standard) was PCR-amplified/mutagenized with primers: phaP10F 5'-GCTTCTAGATGATCCTCACCCCGGAACA-3' phaPSR: 5'-GCTACTAGTGGCAGCCGTCGTCTTCTTTG-3' in order to delete the stop codon. The PCR product was ran on a 1% agarose gel, gel-extracted, cut with XbaI-SpeI and ligated with pSB1AK3 (previously cut with XbaI-SpeI and dephosphorylated). Positive clones were found through digestion screening/sequencing.
When E.coli cells are irradiated with UV light (240 nm), bacteria start synthesizing the fluorescent protein. Why? Eubacteria respond to DNA damage with a set of mechanisms called SOS response. The induction of this system involves more than forty independent SOS genes, most of which encode proteins engaged in protection, repairing, replication, mutagenesis and metabolism of DNA. How does this system work? Under normal conditions, LexA protein represses the transcription of many DNA repairing proteins by binding to some sequences called SOS ???boxes??? in their operators. But, when a damage in the DNA occurs, (different types of genotoxic stress can make this system start), the accumulation RecA-ssDNA-ATP complexes activate LexA for autocleavage.
CheZ is responsible for the dephosphorylation of flagellum-regulating protein CheY. It has been reported that cheZ-/- strain has a higher frequency of direction change and thus a narrower range of mobility.
Cloned from Escherichia coli JM109 strain.
RBS with C-terminal Oligo Arginine - ECFP Fusion
This part has a oligo arginine sequence fused to the C-terminus of an enhanced cyan fluorescent protein. This will be a component of the proof-of-concept part BBa_K331019 which will show localization of proteins into the interior of a microcompartment formed by the assemly of lumazine synthase proteins.
We chose not to include a transcriptional promoter to this part because we wanted the freedom to put it under the control of varied promoters. We also chose not to include a transcriptional terminator to this part because we planned to use it in an operon, where we would be expressing multiple proteins from a single transcript.
This sequence was synthesized by Gene Art
sodA promoter is controlled by some transcription factors including SoxS.
Fumihiko Takeuchi, Aya Iwaki, Akiko Kawashima , Yuki Fukuyama
This it the temperature sensitive cI from the PGW7 plasmid. It has the Elowitz RBS in front (BBa_B0034). It produces a temperature sensitive cI repressor that loses binding efficiency with the cI promoter between 35 and 42 degrees C.
Enzyme in the pathway required for B-Carotene Synthesis. This enzyme is named Geranylgeranyl Diphosphate Synthase. This sequence was taken from a WT strain of xanthophyllomyces dendrorhous. It catalyzes both the conversion of Geranylgeranyl diphosphate to Phytoene and Lycopene to B-Carotene.
Part was PCR amplified from the genomic DNA created from the experiment and paper: High-Level Production of Beta-Carotene in Saccharomyces Cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces Dendrorhous (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1932764/).
Found in the genomic sequence of Xanthophyllomyces Dendrorhous. http://www.ncbi.nlm.nih.gov/nuccore/AY177204.1
HfsG + HfsH proteins from Caulobacter crescentus
Caulobacter crescentus is an aquatic, Gram-negative bacterium that divides asymmetrically and is able to synthetize a strong glue. This glue is mainly made of a polysaccharide. There are different proteins needed to synthetize, export and attach it to the stalk of Caulobacter. To see the hole system, please see this page [1]. In our project, we would like this glue to be produced by Escherichia coli. As E. coli does have homolog genes for many of these proteins, but not for HfsG and HfsH, we decided to create a plasmid including only the genes coding for these two proteins. HfsG is a glycosyltransferase and HfsH [2] is a carbohydrate esterase. Here you have a part made of the Biobricks HfsG (BBa_K196002) and HfsH (BBa_K196003).
As many mutations were needed to make the part compatible with the standard 10, we decided to make it synthetized by GeneArt [3]. We also optimized it for E. coli. We mean that the codons were changed to favour the most present in E. coli.
This sequence comes from Caulobacter crescentus.
branched-chain amino acid transaminase (BAT2); converts leucine to alpha-ketoisocaproate
complete tet./ dox. inducible promoter from Invitrogen vector pcDNA/FRT/TO, T-Rex system
CheZ is responsible for the dephosphorylation of flagellum-regulating protein CheY. It has been reported that cheZ-/- strain has a higher frequency of direction change and thus a narrower range of mobility. With a promoter of your interest, this device rescues the mobility of cheZ-/- cells.
cheZ CDS is cloned from Escherichia coli JM109 strain. Other sequences are from BioBrick parts.
RBS with C-terminal Oligo Arginine - EYFP Fusion
This part has a oligo arginine sequence fused to the C-terminus of an enhanced yellow fluorescent protein. This will be a component of the proof-of-concept part K331019 which will show localization of proteins into the interior of a microcompartment formed by the assemly of lumazine synthase proteins.
TetR regulated by CI operator (RBS+, Term-) with LVA tag
This is a part using the biobrick BBa_K182004 as the upstream part and BBa_S03518 as the downstream part. BBa_K182004 is a cI regulated promoter. The promoter has two binding sites for the cI repressor and is repressed when it binds.It is identical to BBa_R0051 apart from a point mutation in the -35 promoter binding region(15th base is C instead of T). BBa_S03518 is a TetR protein with a Ribosome Binding site upstream and an LVA tag attached to its end. TetR binds to TetR regulated promoters such as BBa_R0040. aTc (anhydrotetracycline) binds to TetR causing it to be released from the promoter and reintroducing transcription. This part comes without a terminator.
BBa_K182004 was used as an upstream part and BBa_S03518 as a downstream part.
This is a BioBrick composed of parts BBa_R0065 and BBa_J06702. This composite produces the mCherry fluorescent protein. It is activated by the presence of LuxR and AHL, and it is repressed by cI.
http://partsregistry.org/wiki/index.php?title=Part:BBa_R0065 http://partsregistry.org/wiki/index.php?title=Part:BBa_J06702
Acyl-ACP Reductase (AAR) - Synechococcus elongatus PCC7942
Produces enzyme that reduces long chain length acyl-ACPs into the corresponding fatty aldehydes
This part codes for the protein LsrK which phosphorylates AI-2 which acts to promote the lsrA promoter.
This sequence is found in the E. coli strain MG1655.
The Ucp1 is a DNA part which encodes for a protein called UCP1 which belongs to the uncuopling proteins family. The uncoupling proteins (UCPs) are transporters, presents in the mitochondrial inner membrane, that mediate a regulated discharge of the proton gradient that is generated by the respiratory chain. This energy-dissipatory mechanism can serve functions such as termogenesis. The protein is regulated by acivation and inhibition in the mitochondrion. The proton conductance in UCP1 is inhibited by nucleotides and activated by fatty acids.
Uncouplin protein UCP1 is a protein that is found in the mitochondria from brown adipocytes.
Lac Promoter/RBS/GeneIII/PhaP/Terminator
Tar receptor under the control of constitutive promoter J23100
The transmembrane chemoreceptor Tar of E. coli mediates chemotaxis towards aspartate. Here we used a set of 8 constitutive [http://partsregistry.org/Part:BBa_J23100 Anderson promoters] from the 2006 Berkeley group to test how different levels of constitutive ''flhDC'' expression affect the motility of ''E. coli''.
* Genomic sequence was amplified using the following primers. ** Primers PCR: Base pairs in caps represent the respective restriction sites followed by either prefix or suffix. Primers were provided by SIGMA. *** Fwd: Tar for + prefix: 5?? actGAATTCgcggccgctTCTAGAtgattaaccgtatccgcgtagtc 3?? *** Rev: Tar rev + suffix: 5?? tcCTGCAGcggccgctACTAGTcaaaatgtttcccagtttggatc 3?? * One XbaI site was found at position 420. The restriction site was changed from TCTAGt to CCTAGt via QuikChange PCR. ** Primers QuikChange (QC): Primers were provided by SIGMA. *** Fwd: TarQC for: 5?? ACTGATTGATTAcCTAGATTATGGCAATACTGGAG 3?? *** Rev: TarQC rev: 5?? TGCCATAATCTAGgTAATCAATCAGTTCAGTTAAC 3?? (the uncapitalized letter induces the mutation for removal of the XbaI site)
*The part was amplified from genomic DNA of ''E. coli'' str. K-12 substr. DH10B, complete genome (CP000948.1). *J23100 information was taken from the partsregistry and physical DNA from the 2012 distribution kit.
RBS (B0034) + MerR (mercury-responsive transcription factor)
This part was designed as a translational unit for MerR expression.
The native RBS of MerR is not strong enough and its intensity can not be predicted as there is no such a corresponding RBS part in Registry. In order to facilitate the use and further characterization of MerR for future team, we prefixed an RBS part BBa_B0034 from Partsregistry.
The coding sequence of MerR comes from Tn, prefixed by an RBS part BBa_B0034 from Partsregistry
The activity of upstream promoter can be qualitatively observed on a X-gal plate, or be quantificationally measured using ONPG assay or fluorescent assay.
A luciferase from Promega's pGL4.10luc2 vector.
sequence of this vector is already published by Promega
pcyA gene transcribed from constitutive promoter.
For the project that this part was made, it was needed to use other Assambly Standard.
CDS was extracted by PCR, promoter and terminator were Classic BioBricks.
IP-Free TannenRFP (Red Fluorescent Protein)
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.
Strong RBS + LacI repressor + double terminator
This part contains a strong ribosome binding site, the LacI repressor and a double terminator (BBa_B0030 + BBa_C0012 + BBa_B0014). This brick comes from the BBa_K292005. The LacI repressor represses pLac promoter from the E.coli lactose operon. So LacI repressor and this bio-brick are really reliable to use when we want to control a gene expression by using pLac promoter. In fact when LacI is not expressed, pLac is activated, and when there is an expression of LacI, pLac is inhibited. We can easily design a system to induce the transcription of LacI by using another operon as the tetracycline operon and creates a failed feedback mechanism for pLac inhibition and so, for gene expression inhibition. The LacI (BBa_K292006) already contains a strong RBS and a double terminator, so this brick is ready to use! History: In E.Coli, pLac is a part of the lactose operon. LacI is constantly expressed and represses the pLac. In the presence of Lactose, LacI is inhibited and pLac is active and allows the transcription of beta-galactosidase. Lactose can be substituted by IPTG (Isopropyl β-D-1-thiogalactopyranoside).
Part:BBa_B0030, Designed by Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003. Group: NCBS, Prather Lab Part:BBa_C0012, Designed by Grace Kenney, Daniel Shen, Neelaksh Varshney, Samantha Sutton Group: Registry Part:BBa_B0014, Designed by Reshma Shetty Group: Registry (2003-07-16)
pCooM Promoter coupled with RBS+RFP+Double terminator
pCooM Promoter coupled with RBS, RFP,double terminator
Device created from a composite of parts.
This antibody detects the espA motif of the typeIII needle complex present in many pathogenic bacteria. This can be surface displayed for pathogen detection applciations.
gene synthesized, sequence taken from La Ragione, R et al. Recombinant anti-EspA antibodies block Escherichia coli O157:H7-induced attaching and effacing lesions in vitro. Microbes and Infection. September 2005; 8: 426???433. Available Online: http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6VPN-4H4T215-1-7&_cdi=6211&_user=4420&_orig=search&_coverDate=02%2F28%2F2006&_sk=999919997&view=c&wchp=dGLbVtz-zSkzS&md5=f35699db5dcd202f4cd1c1d8739d89b0&ie=/sdarticle.pdf
2-oxoglutarate oxygenase/decarboxylase (EFE)
this part is laci-cfp fusion protein. laci sequence was obtained from pubmed and was synthesized.
we followed up all the parameters needed to make it as a registry part.
pBADcIT-1.0:cI protein expression system activated by arabinose(RBS strength:1.0)
Sifan Wang,Yizhen Yan,Jianzhao Chi,Qingshu Wu
OmpR234 protein, with higher effect on Curli promoter
This part is coding for a mutated protein OmpR. This protein will be phosphorilated by the complex Envz. The phosphorilated protein is an activator of the Curli promoter
Tet Repressible C-terminal Arg Tagged ECFP (no terminator)
This is BBa_K331025 with the tetracycline repressible promoter BBa_R0040 added in front. This will be a component of a proof-of-concept part that will show localization of proteins into the interior of a microcompartment formed by the assembly of lumazine synthase proteins.
Arabinose Promoter with Tetracycline Inverter
BBa_E1010 with an LVA tag added via PCR.
This composite part contains LacIQ (BBa_I14032) and Estrogen Receptor Element (ERE) and TetR (BBa_C0040). The Estrogen Responsive element is to be used in conjunction with ER (Estrogen Receptor). This design involves ER binding to the ERE and disrupting the constituative promoter LacIQ from producing TetR via physically getting in the way of the polymerase.
There is some data that suggests the ER is toxic to the cell. However in our experiments while we did see a decrease in growth rate we did not find ER to be fatal to the cell.
The Estrogen Responsive Element was taken from the Human Estrogen Responsive Element
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).
RFP Coding Device - RFC10 and RFC25 compatible
Red Fluorescent Protein from Discosoma striata BBa_E1010
optimized sfGFP with Csy4 insulator loci
Csy4 is a member of CRISPR pathway discovered in Pseudomonas aeruginosa. It is a single endoRNase that recognizes and cleaves a 28-nucleotide repetitive sequence and produces stable transcripts with a 5???hydroxylgroup, which can eliminate unwanted interactions between 5???UTRs and translational elements such as RBSs to standardize the expression of the elements.
Csy4 is a protein used to cleavage the mRNA so that the influence from UTR will be eliminated
The Csy4 part is modified from the plasmid pHMGWA-Pa14Csy4 made by Jennifer Doudna's Lab.
This photoreceptor was assembled by Strickland et al., and consists of a LOV (light-oxygen-voltage) domain of Avena sativa phototropin1 (AsLOV2) that senses blue light, fused to the trpR- DNA binding domain of the transcription factor trp repressor. The resulting protein is named LovTAP: LOV- and tryptophan-activated protein. LOV domains bind a flavin-mononucleotide (FMN) or flavin-adenine-dinucleotide (FAD) cofactor, which are used in a wide variety of metabolic pathways as cofactors in redox reactions and are available in most organisms. The cofactor has a broad absorption spectrum, with a maximum at 450 nm. Besides, the core of the LOV domain is often flanked by amino- or carboxy-terminal helices, termed A???α and Jα, respectively In the LovTAP construction, AsLOV2 domain via its carboxyl-terminal Jα ???helix was ligated to an amino-terminal truncation of TrpR. The resulting protein has a domain-domain overlap with a shared helix. Thus, photoexcitation would change the conformation of the protein, in turn changing the stability of the shared-helix-domain contacts. Under the presence of light, absorption of a photon leads to the formation of a covalent adduct between the flavin mononucleotide (FMN) cofactor and a conserved cysteine residue in the AsLOV2 domain, which results in conformational rearrangements in LovTAP. This change impacts the affinity of the shared helix for the two domains: disrupting the contacts between the shared helix and the LOV domain and enabling the association of the shared helix with the TrpR domain, which establishes DNA-binding affinity and LovTAP can then bind DNA as an homodimer, repressing the transcription of the genes downstream of its binding sites. In the dark, when the shared helix contacts the LOV domain, the TrpR domain's DNA-binding affinity decreases and LovTAP is in an inactive conformation.
We decided to synthesize a new LovTAP part, that in comparison with the Part:BBa_K191003 that is already at the registry, has the following differences: 1. The 2 PstI restriction sites were removed from the coding region of LovTap. 2. We included a punctual mutation to change the ILE427 by a PHE427, as was proposed by the model results of the team iGEM09_EPF-Lausanne. With this mutation LovTAP should react faster and the conformational change should be more stable (the protein stays in the active form for longer, under light induction). 3. The part does not include a promoter. We eliminated the inverting regulator sensitive to LacI and CAP protein(Part:BBa_R0010). According to the report of EPF-Laussane team, the expression levels of LovTAP under the inverting regulator, doesn??t seem to show differences to the induction with IPTG. Thus, we decided to remove it and as we know that the level expression of LovTAP must be low (because at high levels of expresion, there is unspecific regulation under darkness and light states), we plan to test weak promoters to choose the best option and include it in the construction. 4. The RBS was changed from a strong (Part: BBa_B0030) to a medium strength (Part:BBa_B0032), thus expecting lower levels of lovTAP protein. 5. The stop codon tga was changed for two taa.
Chiameric protein assembled by Strickland et al. Reference: Strickland, D., Moffat, K., & Sosnick, T. (2008). Light-activated DNA binding in a designed allosteric protein. Proceedings of the National Academy of Sciences, 105(31), 10709. National Acad Sciences. Retrieved from http://www.pnas.org/content/105/31/10709.full.
PrsaA constitutive promoter, esp optimized for Caulobacter, and rsaA
PrsaA constitutive promoter from Caulobacter crescentus, esp, a suspected serine protease from Staphylococcus epidermidis, optimized for expression in Caulobacter crescentus, and the C-terminal secretion tag from rsaA.
Stop codon naturally occurring at the end of esp removed to ensure smooth expression of the fusion protein.
PrsaA and rsaA C-terminal secretion tag both come from the genome of Caulobacter crescentus; esp optimized for Caulobacter crescentus is a synthetically codon optimized copy of esp as it is found in Staphylococcus epidermidis.
cyan fluorescent protein from PamCyan-1C1
Aldehyde Decarbonylase (ADC) - Synechococcus elongatus PCC7942
Produces the enzyme that removes the carbonyl group (C=O) from the fatty aldehyde, yielding an alkane one carbon shorter than the original Acyl-ACP and a molecule of formate
Aldehyde decarbonylase gene from synechococcus elongatus
When there is no ammonia in the media the RNA-polimerase binds to RpoN Promoter and the transcription starts. Why? In gram-negative bacteria, transcriptional activation in response to some external stimuli (absence of nitrogen, for example) often involves the alternative sigma factor, Sigma 54. This factor, also called RpoN or Sigma N, was originally identified as the sigma factor for nitrogen-controlled genes. Sigma 54 works in conjunction with members of the NtrC (Nitrogen regulatory protein C) superfamily of transcriptional activators. ???RpoN Promoter??? contains a binding site which is recognized by the RNA-polimerase when Sigma 54 is binding to this protein. The polimerase binds to RpoN promoter which is present on genes/operons whose products minimize the slowing of growth under nitrogen-limiting conditions (aprox. 2% of the E. coli genome appears to be under NtrC control).
We just combine part J01003 with part I13521, J01003 is coden oriT and can transfer the sequence that followed.
This part can just be moblized by other plasmids which contain tra gene.
Both the two parts we used were from registry.
This is the ''virG'' response regulator from the virA/G receptor system. The virA/G receptor system is used by ''Agrobacterium tumefaciens'' to detect phenolic substances secreted by plants. In presence of these substances, ''virG'' is activated by the ''virA'' receptor and induces the transcription of genes under the control of a ''vir'' promotor. Normally, ''virG'' needs the ''rpoA'' RNA polymerase subunit from ''A. tumefaciens'' to work in ''Escherichia coli'' but this ''virG'' BioBrick is mutated, so works with the ''rpoA'' subunit from ''E. coli''.
synthesized by Mr. Gene, so optimated codon usage, removal of illegal restriction sites
LacI (Lva-, N-terminal deletion) regulatory protein
LacI is a regulatory protein responsible for the repression of many catabolite genes. Transcription is regulated by proteins which bind operator sequences around the transcription start site. These proteins can positively affect transcription (activators) or negatively affect transcription (reppresors). Some repressor proteins can be inactivted however by addition of an inducer, such as IPTG or certain sugars. LacI if the regulator protein for the lactose operon in ''E.coli'' and the hyper-spank protein of ''B. subtilis''#1( BBaK143015 ) and is responsible for ensuring that in the absence of lactose (or IPTG) that there is no expression trough these promoter. LacI is not endogenous to ''B. subtilis'', so LacI will need to be expressed in the host in order for the hyper-spank promoter to be regulated. In the presence of IPTG or lactose, the LacI tetramer is unable to bind DNA and so transcription resumes. This version of LacI lacks a Lva degradation tag and has a small(3 amino acid) N-terminal deletion relative to the current registry LacI ( BBa_C0012</bbpart)> and is derivatives. The N-terminal deletion appears to be common to most of the LacI genes used in conjunction with ''B. subtilis'' though both forms are found in ''E.coli'' (in differing strains). LacI was used in conjunction with the '''Hyper-spank promoter''' ( BBa_K143015 ) and acted as an input adaptor for a '''Polymerases per second''' (POPS) output ====References==== #1 pmid=16166525
LacI was located in the sequence of the ''B. subtilis'' shuttle vector pDR111. This version of LacI lacks a Ltva degradation sequence and has a small N-terminal deletion that is observed in many LacI used in studies on ''B.subtilis''. In particular, this LacI protein is used in pDR111 to regulate expression of the inducible Phyper-spank protein ( BBa_K143015 ) (also used in the pDR111 vector). The BioBrick prefix and suffix were applied to the gene
The LacI gene was cloned from''B. subtilis'' shuttle vector pDR111 using Pfu DNA polymerase PCR
FFT (BBa_K899001) with double RBS (BBa_K899009)
This is a composite part containing our fructan:fructan fructosyltransferase (FFT) gene with a "double" RBS containing both the shine-dalgarno and kozak sequence. This is the main part of our project, and also what we submitted to iGEM. The part was characterized in a pJET vector (http://www.bioinfo.pte.hu/f2/pict_f2/pJETmap.pdf) and we found a weird looking substance that resembled sugar structure in the growth medium. When we expressed FFT in E. coli top 10 strains they grew as fast as regular top 10 bacteria, probably because the sucrose:sucrose fructosyltransferase (SST) enzyme wasnt present (this enzyme produces the base that FFT polymerizes). When staining for inulin we observed a slight increase in inulin even though the SST enzyme wasn't present. This is probably because there was something in the substrate that enabled FFT to polymerize the sugar.
The FFT gene derives from the jerusalem artichoke (Helianthus Tuberosus)
This is the part we actually created to test the glue production by E. coli. HfsG and HfsH come from Caulobacter crescentu. Caulobacter crescentus is an aquatic, Gram-negative bacterium that divides asymmetrically and is able to synthetize a strong glue. This glue is mainly made of a polysaccharide. There are different proteins needed to synthetize, export and attach it to the stalk of Caulobacter. To see the hole system, please see our wiki page [1]. In our project, we would like this glue to be produced by Escherichia coli. As E. coli does have homolog genes for many of these proteins, but not for HfsG and HfsH, we decided to create a plasmid including only the genes coding for these two proteins. HfsG is a glycosyltransferase and HfsH is a carbohydrate esterase. RFP has been added as a reporter.
As many mutations were needed to make the part compatible with the standard 10, we decided to make hfsG and hfsH synthetized by GeneArt [2]. We also optimized it for E. coli. We mean that the codons were changed to favour the most present in E. coli.
HfsG and HfsH sequences come from Caulobacter crescentus.
The aequorin-coelenterazine complex needs calcium to produce light. In the process this calcium enters through a special type of calcium channels present in the cell???s plasma membrane which open or close in response to a change in the transmembrane potential, that is why they are called voltage-dependent calcium channels (VDCC). The equation which describes calcium current through these channels is the following: Where g is the conductance associated with the channel, V is the transmembrane potential and ECa is the Nernst potential, related to the different ionic concentration inside and outside the cell. Considering that these channels are only permeable to calcium and have two states -open or closed-, the total conductance associated with the population of VDCCs can be expressed as the maximal conductance ( ) times the fraction of all channels that are open. This fraction is determined by hypothetical activation and inactivation variables m and h, which depend on voltage and time: VDCCs are well characterized in excitable cells such as neurons, where the value of is known: Budding yeast, Saccharomyces cerevisiae has homologous voltage-dependent channels in its plasma membrane. However, there is a lack of study of the properties of these channels. That is why we have determined their conductance according to our experimental results: Having characterized calcium entry to the cell, we studied the aequorin production of light through a calcium-mediated response after two types of stimulation: ??? Chemical stimulation Alkali shock (performed by the addition of KOH) induced a calcium entry to cytoplasm (where aequorin is located) through voltage-dependent calcium channels. These calcium ions bind the aequorin-coelenterazine complex and photons ???light- are emitted during a short period of time. This graph shows yeasts??? response to the addition of 30 ??L of KOH: Furthermore, we made experiments with increasing KOH volumes (in microliters) and realized that light emission was higher: the more KOH arrives to cells, the more calcium ions get into the cytoplasm and more photons are emitted by aequorin. It has to be noted that, in presence of suitable amounts of coelenterazine, the aequorin production of light can be repeated with several stimulations of 30 microliters each (where arrows show): Finally, we have proved that calcium enters through VDCCs, as yeast strains lacking this calcium channels (cch1 and mid1) emit less amount of light. Two negative controls (addition of EDTA, a quelant of calcium ions, and KCl, which do not perform alkali shock) are also included in this graph: ??? Electrical stimulation Calcium entrance to cytosol ???and the consequent aequorin light emission- can also be perfomed by applying electricity to the yeast culture. This response was characterized at different voltage values and times of application. Again, the stimulation was repeatable (stimulus applied where arrows): *The following graph show data obtained with an ELISA plates reader which made discontinuous measures, one each 30 s. Therefore, we had not information about light production between dots. The time of exposure to the electrical stimulus was crucial, even more that the aplied voltage. That means, if we increased the voltage at very short times, cells could produce a more abrupt peak of light. But if we increased the time of exposure to the electricity, we observe a less defined response, with more flattened peaks. That???s probably because a big exposure time of electrical input damages and kills the yeasts, making them to release their components to the medium, including the aequorin-coelenterazine-Ca2+ complex, so the emission of light is more uniform in time, instead of the production of the flash produced by the Calcium entering in the cell. In the case of very little voltages (like 1,5V) this observation is not carried out by our yeasts. The reason must be that the electrical input is too low, so yeasts don???t die so easily as with more elevated voltage, and a better response is produce with a more prolongated electrical shock. Although the previous graphs show that the second stimulation induces a lower amount of light, varying the time of stimulus application, higher peaks could be obtained: Similarly to chemical stimulation, yeast mutant strains lacking calcium channels had less light emission. Several negative controls (water, SD medium, SD with coelenterazine without yeasts, yeasts without coelenterazine, and addition of EDTA) were made to prove that light came from aequorin-transformed yeasts: *The following graphs show data obtained with a continuous luminometer. It has to be noted the sharp peak of light produced when stimulation is applied (we were not able to detect it with the discontinuous measurements) In the following graphic WT aequorin-transformed yeasts and mutant strains (lacking cch1 and mid1) are compared again. SD medium with coelenterazine and yeasts without coelenterazine are included as negative controls: Again, light production is lower in mutant cells, also in the first peak of light -that we were not able to see with the discontinuous luminometer-, as calcium ions enter through VDCCs. A sharp peak of light is produced when the stimulus is applied, but our controls discard the idea of an artifact. For example, light could be made by a spark produced during the discharge. It is not very probable, because the peak observed was produced during near 400 seconds after the stimulus. Another time, when cells without coelenterazine or mutants were used, we saw no light.
It comes from the genomic sequence of the luminiscent jellyfish Aequorea victoria.
J23117 constitutive promoter + LovTAP Photosensor
Optimized esp gene for C. crescentus
The wild Type esp is secreted by S. epidermidis, a bacteria that has low G/C content in its genome. It is then optimized to give a high G/C content without changing any amino acids sequence.
DNA codons are optimized for C. crescentus expression.
Wild type esp sequence from Zhang,Y.Q., Ren,S.X., Li,H.L., Wang,Y.X., Fu,G., Yang,J., Qin,Z.Q., Miao,Y.G., Wang,W.Y., Chen,R.S., Shen,Y., Chen,Z., Yuan,Z.H., Zhao,G.P., Qu,D., Danchin,A. and Wen,Y.M. Genome-based analysis of virulence genes in a non-biofilm-forming Staphylococcus epidermidis strain (ATCC 12228). Mol. Microbiol. 49 (6), 1577-1593 (2003). Optimized sequence is based on the wild type sequence.
Combines BBa_B0034 RBS (Elowitz 1999) with BBa_J06601 mCherry with forward terminator
Test constructs involving this part had no fluorescence observed. Sequencing returned nothing resembling the expected part.
pLac+RBS+Secretion Signal and Streptavidin Binding Tags
CFP under the control of a CMV promoter.
This part was split from its plasmid backbone BBa_K157040.
EGF-Receptor_3rd domain with PelB leadersequence, His-tag and C-terminal Sortase motif
Estrogen receptor refers to a group of receptors that are activated by the hormone 17β-estradiol(estrogen). member of the nuclear hormone family of intracellular receptors.
The reconstruction of the DBD to the hinge region, LBD and Backbone will be done with RFC 25
Human ER (of the nuclear hormone receptor superfamily)
PhytoBricks parts from plasmid pUAP78141
heat sensitive cI QPI with high promoter
This is a heat sensitive cI QPI with a high promoter preceding the repressor (BBa_J23114). It can be used to construct a heat-inducible system if a reporter or indicator is adding to the right of this part.
This is a mutagenized form because the original product had an extra PstI site between the repressor and terminator. While there are still extra basepairs between the end of the repressor sequence and the Biobricks scar, they no longer code for a PstI site. The extra base pairs are: [repressor coding region] CTCCAGCGGCCGC [Biobricks scar]
PGW7 plasmid and various BioBricks parts.
Yellow fluorescent protein mCitrine. ===See also=== * BBa_J18930 ===References=== * Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY. (2001) Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. PMID: 11387331
* starts with consensus VSKGEEL * ends with consensus DELYK
Mutate seven phosphorylation site of Sic1, stopping the cell cycle in the G1 phase.
Mutate seven phosphorylation site of Sic1 and one site of Spe1's restriction site.
TNT-R3: a computationally designed receptor binding TNT, made by mutation of the Escherichia coli periplasmic ribose-binding protein: see Looger, L., Dwyer, M.A., Smit, J.J., and Wellinga, H.W. 2003. Computational design of receptor proteins with novel functions. Nature 423, 185-190. This receptor interacts with a hybrid transmembrane protein Trz, created by combining the normal partner of ribose-binding-protein, Trg, with the intracellular part of 2-component sensor protein EnvZ, which normally phosphorylates response regulator protein OmpR, allowing it to activate promoters such as PompC (see Baumgartner, J.W., Kim, C., Brisette, R.E., Inouye, M., Park, C., and Hazelbauer, G.L. 1994. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg thate recognizes sugar-binding proteins to the kinase/phosphatase domainof Osmosensor EnvZ. J. Bacteriol. 176, 1157-1163). To use this receptor, include it in a system with Trz and a reporter gene controlled by PompC.To avoid cross-talk, it may be necessary to use a chassis lacking EnvZ and ribose-binding protein.
Derived by mutagenesis of the Escherichia coli periplasmic ribose-binding protein: see Looger, L., Dwyer, M.A., Smit, J.J., and Wellinga, H.W. 2003. Computational design of receptor proteins with novel functions. Nature 423, 185-190.
Cinnamoyl-CoA reductase from Arabidopsis thaliana under pTET constitutive promoter
* first fragment of the split enzyme beta-lactamase * NgoMIV / AgeI protein fusion part * iGEM Team Freiburg 2007
between BioBrick 1.0 sites part is flanked 5' by NgoMIV and 3' AgeI(=PinAI) to facilitate in frame cloning for protein fusions
Keratinocyte growth factor (KGF) is a locally acting epithelial mitogen that is produced by cells of mesenchymal origin and has an important role in protecting and repairing epithelial tissues.Use of recombinant human KGF (palifermin) in patients with hematologic malignancies reduces the incidence and duration of severe oral mucositis experienced after intensive chemoradiotherapy. These results suggest that KGF may be useful in the treatment of patients with other kinds of tumors, including those of epithelial origin.[1]The potential effect of KGF on wound healing was assessed in vitro by measuring randomized migration and plasminogen activator (PA) activity of keratinocytes in response to the growth factor. Incubation of normal human keratinocytes with KGF in modified MCDB 153 medium significantly stimulated cell migration[2] The Keratinocyte Growth Factor (KGF), also known as FGF7, is a growth factor present in the epithelialization-phase of wound healing. In this phase, keratinocytes are covering the wound, forming the epithelium.[3] . KGF is weakly expressed in human skin, but is strongly upregulated in dermal fibroblasts after skin injury. Its binding to a transmembrane receptor on keratinocytes induces proliferation and migration of these cells. Furthermore, KGF has been shown to protect epithelial cells from the toxic effects of reactive oxygen species. We have identified a series of KGF-regulated genes that are likely to play a role in these processes. In addition to KGF, activin seems to be a novel player in wound healing.[4]
The gene is in pMA vector having biobrick restriction sites(Ecor1, Xba1 and Spe1,Pst1,respectively)
AlnA is a protein with emulsification activity originated from the natural oil degrading bacterium Acinetobacter radioresistens. It has been shown that this protein keeps its emulsifying capacitiesis when it is produced by other organisms, such as Escherichia coli. AlnA has an amphipathic structure, which allows it to form micelles that accumulate at the interface between liquids of different polarities such as water and oil. This process is based upon the ability of biosurfactants to reduce surface tension, blocking the formation of hydrogen bridges and certain hydrophilic and hydrophobic interactions. The emulsifier helps to disperse the oil by emulsifying the oil, thus increasing the surface area for the growth of microorganisms on hydrocarbons
Sequence from Tsien's lab; Code for the expression of Infrared protein; must co-express with Heme Oxygenase-1 (HO1) to produce infrared signal
PoPS -> PhiR73 pag - PO promoter -> PoPS
Louise Linnebjerg Bohn Christoffersen, Pernille Marie Madsen, Sheila Maibom-Thomsen
This part entails a sigma 70 promoter, a RBS, the FlhDC master operon and a dual-terminator. This part will cause hyperflagellated cells.
The promoter, the terminater and the RBS are all in the spring 2010 kit. The FlhDC mater operon is fron E. coli.
CooA is a heme-containing transcriptional activator that enables Rhodospirillum rubrum to sense and grow on CO as a sole energy source. CooA is a member of a family of transcriptional regulators similar to the cAMP receptor protein and fumavate nitrate reduction from Escherichia coli. The protein is active in sequence-specific DNA binding in the presence of CO, but not in the absence of CO. The protein to be a dimer in the absence of CO. The product, CooA, is 28% identical (51% similar) to CRP(cAMP receptor protein) and 18% identical (45% similar) to FNR(fumavate nitrate reduction) from Escherichia coli. Inactive Fe(II) CooA structure adapted from that of the strain with PDB identification no. 1FT9. The protein consists of two monomers, shaded differently, which dimerize along the central C-helices of adjacent effector-binding domains. The solved structure is asymmetric, in which one monomer contains fused C- and D-helices. Nonetheless, both F-helices that interact with DNA in a sequence-specific manner are buried from the surface in the structure. The 4/5 loop is noted and so are the Pro2 and His77 heme Fe(II) ligands.
The sequence information was acquired from NCBI and physical DNA was synthesized from GENEART. Classical cloning strategies(restriction digestion) were used to produce biobricks.
Hwan Youn, Robert L. Kerby, Mary Conrad, et al. 2004. Functionally Critical Elements of CooA-Related CO Sensors. J. Bacteriol. 186(5):1320-1329. doi:10.1128/JB.186.5.1320-1329.2004.
This is the Composite Part which creates the enzyme Lumazine Synthase. Lumazine SYnthase generates the product Lumazine which aggregates together to form a microcompartment, or synthetic organelle. The microcompartment has large negatively charged pores through which proteins containing positely charged Arginine Tags may be targetted.
point mutations to EcoRI and PstI sites?
Contains an IPTG inducible promoter an RBS, RFP, and terminator.
cjBlue is a blue (λ[maxabs]=610nm) chromoprotein from the sea anemone Cnidopus japoincus, as reported by (Chan et. al., 2006). We synthesized cjBlue codon optimized for expression in e.coli and observed a turquoise colour, wavelength approximately 5*10^2nm. This part is also submitted as a functionally active variant with J23110 and B0034
This chromoprotein from the coral Stylophora pistillata, spisPink (also known as spisCP), naturally exhibits strong color when expressed. The protein has an absorption maximum at 560 nm giving it a pink color visible to the naked eye. The strong color is readily observed in both LB or on agar plates after less than 24 hours of incubation. The protein spisPink has significant sequence homologies with proteins in the GFP family.
Stylophora pistillata. The protein was first extracted and characterized by Alieva et. al. under the name spisCP (GenBank: ABB17971.1). This version is codon optimized for E coli by Genscript.
cI sequence slightly different from part C0051. NCBI locus: NC_001416
This part is assembled with pBad promoter, rbs.pelB> (pelB prepro with native rbs and start codon, but no stop), and a <phoA> region (no start or stop codon). ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
This part was made using AKL41, which consisted of {Pbad}.{rbs_pelB>}.{<phoA!}.{<b1006>}. The stop codon of the phoA region and the <b1006> terminator were removed. PCR with ca998 Forward oligo, and mea37 reverse oligo for {<phoA>}.
This part was made by ligating I13025 to B0015.
These parts can be found in the registry.
This part (submitted in pSB1A2) consists of the lac promoter and lacZ' gene, encoding the N-terminal 76 amino acid residues of LacZ, sufficient to complement the lacZ-delta-M15 mutation for blue-white selection on Xgal plates. A SacI site has been introduced at the 3' end, overlapping the XbaI site of the Biobrick prefix. This is designed to be used as a cloning vector for making new biobricks. PCR primers can be designed with a SacI site in one primer and an SpeI site in the other. This removes the necessity for an excessively long non-complementary tail on one primer bearing either the full biobrick prefix or suffix. The PCR product can then be digested with SacI and SpeI for insertion into this plasmid, replacing the Plac-lacZ' cassette. Recombinant plasmids will then be white on IPTG/Xgal plates, whereas any that still contain the original insert will be blue. We have used this strategy to prepare several biobricks, including BBa_J33204, which contains the xylE gene encoding catechol-2,3-dioxygenase. (For making biobricks that contain lacZ', BBa_J33204 can be used in the same way; in this case, clones with plasmids that still contain xylE will turn yellow on addition of a drop of 10 mM catechol.)
Note that the SacI site overlaps the SpeI site. The Biobrick prefix ends ...TCTAGAG. When this is added to the CTC at the start of the sequence shown here, the SacI site, GAGCTC, is generated.
The DNA was amplified from E. coli BL21 genomic DNA using primers based on published sequence (Genbank accession J01636, gi:146575). The annotation shown here is based on that associated with this Genbank entry. The sequence shown here is derived by sequencing the construct.
Constitutive expression cassette for TetR +LVA (J23100.B0034.C0040.B0015)
The Biobrick encodes tetR (BBa_C0040) under control of the constitutive promoter (BBa_J23100) followed by the ribosome binding site BBa_B0034. The transcription of tetR is terminated by the double terminator BBa_B0015
Cinnamoyl-CoA reductase from Arabidopsis thaliana under arabinose promoter
This part is a delta-15 desaturase derived from Synechocystis sp PCC 6803. The enzyme is able to introduce a double bond at the Δ-15 site in the hydrocarbon chain of linoleic acid (18:2 ; Δ9,12), for its conversion into alpha- linoleic acid (18:3 ; Δ9,12,15), an Omega-3 polyunsaturated fatty acid (PUFA). Mass spectrometry results show that when feeding oleic acid to E. coli transformed with this part in addition to delta-12 desaturase (BBa_K925000), alpha-linoleic acid is observed in membrane lipids.
RFP-BA-BbsI (for construction of BA BioBytes 2.0)
Test plasmid for the construction of new Bytes-v2. The RFP cassette can be removed using the BbsI enzyme and a new ORF or cassette may be inserted into the plasmid allowing for testing of the part with the BioBytes Assembly System-v2.
In wild type E. coli, ClpX forms a ring of hexamer and binds to a double heptamer of ClpP. ClpX recognizes a specific C-terminal degradation tag called SsrA and starts to unfold the tagged protein. The denatured polypeptide is translocated into the ClpP subunit, which breaks peptide bonds. The N terminal end of ClpX contains a domain which is responsible for interaction with its natural adaptator protein (SspB) that we would avoid. As this N-terminal domain is also partially responsible for ClpX subunits complexation into an hexamer, fusing three C-terminal end of ClpX together with appropriate linkers increases the stability of the system (T. Baker et al.) in the absence of this N-terminal domain.
We deleted 2 EcoRI sites and 2 Age1 sites by directed mutagenis at positions 522 and 967, and 183 and 921 respectively. This biobrick has standard RFC-25.
LovTAP: Blue Light Photosensor and Transcriptional Regulator
Fusion protein of BisdA and BisdB behind constitutive promoter
This is a reporter for use in a C. elegans chassis. Excitation wavelength of 434 nm and emission wavelength of 477 nm.
Genetic mutant of GFP (Green Fluorescent Protein) from Aequorea victoria. (pIC517).
Fusion EYFP with C-terminal Arginine Tag
This Yellow Fluorescent protein has been fused with 10 C-terminal Arginines to target it into the Lumazine-based microcompartment.
BioBrick Foundation EYFP fused with a synthesized Arginine tag
B. subtilis levansucrase. Lethal to E. coli in presence of sucrose.
sacB encodes the Bacillus subtilis levansucrase,which is the enzyme catalyzing hydrolysis of sucrose and synthesis of levans(high-molecular-weight fructose polymers). It is lethal to gram-negative bacteria E-coli. It works with cat as an alternative method for inserting BioBricks into the genome by using homologous recombination instead of restriction digestion, with the added bonus of not leaving a marker behind in the product. Protocol is shown here: http://2010.igem.org/Team:Edinburgh/Project/Protocol
Note: MABEL was used to mutate an internal EcoRI site to GAGTTC (underlined). Features: coding sequence runs from 13 to 1434, RBS 1 to 3, EcoRI site mutated 211 to 216.
Source: Bacillus subtilis 168 genomic DNA. Sequence (from SacBPlan11June10.docx)
Continuous expression of LasR receptor protein (with RBS, B0030)
Optimal strength of constitutive promoter.
Composite part consisting of individual parts in the registry.
IPTG inducible Cysteine desulfhydrase (CysDes)
This composite part was designed to construct a working CysDes gene under the control of an inducible promoter: the presence of IPTG inhibits the LacI repressor protein binding to the LacO operator; in this way it allows the RNApol to bind to the Ptac promoter and trascribe the gene of interest. In this case the gene of interest is a Cysteine Desulfhydrase, an aminotransferase that converts cysteine into pyruvate, ammonia and hydrogen sulfide.
LacI+LacIq promoter is an autonomous part (K731300) also designed by our team.
LacI+LacIq promoter comes from pET-21b plasmid. CysDes gene comes from the genome of Treponema denticola.
Constitutive promoter and RBS BBa_K081005 + Caulobacter optimized esp + C-terminal sec
Produces and secretes active Esp with intact C-terminal secretion tag from RsaA. Esp is a suspected serine protease that breaks up Staphylococcus aureus biofilms.
Optimized esp does not have a stop codon to ensure expression of the fused secretion tag.
BBa_081005 was submitted to the registry by 2008 iGEM Team UNIV_Pavia, codon optimized esp was synthesized by Integrated DNA Technologies, rsaA C-terminal secretion tag was copied from Caulobacter lab strain CB15N.
tetracycline resistance protein TetA(C) (backwards) [cf. BBa_J31007]
This is the coding region for Tetracyline resistance in the reverse orientation. If a promoter adn RBS are placed downstream and the entire region, from promoter to stop codon is flipped the cell will express tetracycline resistance.
LuxS is a synthase for the AI-2 quorum sensing system. This protein is involved in the production of small molecules that are readily absorbed by multiple species of bacteria. LuxS can be used as part of a positive feedback loop in cell to cell signaling.
Nucleotide position 54 changed from A to T in order to destroy a PstI site; this is a sense mutation Also changed stop codon from TAG to TAA (recommended in Registry Help)
The LuxS sequence was pulled from E. coli MG1655 by predesigned primers.
AI-1 generater p(tetR)+RBS+LasI+terminater
The M.ScaI protein is a type II methyltransferase (subtype beta) that recognises site on the DNA of the following sequence 5..AGTACT..3. It methylates this site at the 5th (Cytosine) nucleotide leaving ao an N4-methylcytosine (m4). This methylation type (m4) is not found in native E. coli nor is the recognition site methylated by any of E. coli's native methylation systems (Dam, Dcm). This part also includes a flexible linker with incorporated myc-tag at the N terminus of the protein. This will allow for easier creation of fusion proteins and expression verification using westerblot (using the myc antibodies). It can be assumed that M.ScaI (with linker) is expressed and folds properly in E. coli because its function has been verified by the iGEM Amsterdam 2012 team.
Contains no forbidden sites (according to the RFC-10 protocol). Contains no sites vulnerable to E. coli's native restriction systems.
Natively found in Streptomyces caespitosus. Sequence obtained from REBASE. Synthesized by IDT.
Fructan fructan fructosyltransferase (FFT)
Elongates a trisaccharide with the fructose unit of a sucrose substrate into an inulin polymer. Works together with part BBa_K899000 (SST).
7 BP from start codon we embedded an shine dalgarno sequence.
Extracted from Jesrusalem Artichoke (helianthus tuberosus)
aTc inducible system (aTc->PoPS) with J23100 promoter
Lorenzo Pasotti, Letizia Diamante, Paolo Magni
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).
We have attached the cI insert to a terminator vector, which made the next step construction more convenient.
The length of terminator part is quite small, so it should be digested to a vector and attached to the cI insert.
Built by joining different standardized biobricks together
pBADcIT-0.07:cI protein expression system activated by arabinose(RBS strength:0.07)
Sifan Wang,Yizhen Yan,Shuqin Hu,Yunxin Long,Zhao Ma
A protein from Chlamydomonas sp. W80 that increases the salt tolerance of E.coli. This protein is called bbc1. The exact mechanism of the increased salt tolerance is as of yet unknown. But since bbc1 shows a high homology to RBS binding proteins, it is theorized that it may prevent/stabilize the folding structure of the ribosome under high salt stress conditions.
This part is oriented opposite to typical BioBrick arrangement in terms of placement of BioBrick prefix and suffix. This part can be ligated to a promoter to measure promoter activity.
A more sensitive oxygen detector in bioreactor. Constructed with mNark promoter and enhanced YFP with LVA tag.
and hence also stronger activity under anaerobic conditions. It is also shorter than the NarK promoter (BBa_K239005)
mNarK promoter is designed to have better affinity to activated Fnr than the NarK promoter (BBa_K239005) and hence also stronger activity under anaerobic conditions. It is also shorter than the NarK promoter (BBa_K239005).
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
The ecdysone receptor is a nuclear receptor found in arthropods, where it controls development and contributes to other processes such as reproduction. The receptor is a non-covalent heterodimer of two proteins, the EcR protein and ultraspiracle protein (USP). It binds to and is activated by ecdysteroids. Pulses of 20-hydroxyecdysone occur during insect development, whereupon this hormone binds to the ecdysone receptor, a ligand-activated transcription factor found in the nuclei of insect cells. This in turn leads to the activation of many other genes,WHICH Ultimately causes physiological changes that result in ecdysis (moulting).
Human EcR (of the nuclear hormone receptor superfamily)
This is the coding sequence of superfolder GFP (Pedelacq et al (2006): "Engineering and characterization of a superfolder green fluorescent protein", Nature Biotech 24 (1) January 2006). It carries the following amino acid changes with respect to mut3 GFP (E0040), the currently most commonly used GFP in the registry: S30R, Y39N, F64L, G65T, F99S, N105T, Y145F, M153T, V163A, I171V, A206V Its in-vivo properties are considerably improved with respect to mut3 - it develops fluorescence about 3fold faster than mut3 GFP and reaches 4fold higher absolute fluorescence levels. Fluorescenct colonies can be identified with the naked eye even without UV or blue light illumination (that is to say the amount of blue light in normal daylight or lablight is sufficient). Additionally it is more stable in vitro and refolds faster after in vitro denaturation with respect to mut3 GFP. Note: Superfolder GFP is available in constructs driven by the pBAD and T7 promoters: part numbers I746908 and I746909 respectively. Additionally 6-his tagged versions for protein purification exist: I746914 (pBAD driven) and I746915 (T7 driven).
Codon optimisation before de novo synthesis was carried out for both, E.coli and Bacillus subtilis.
Superfolder GFP was originally described by: Pedelacq et al (2006): "Engineering and characterization of a superfolder green fluorescent protein", Nature Biotech 24 (1) January 2006 This version was synthesised de novo (by Geneart).
endo-beta-1,3-1,4 glucanase (BglS) from Bacillus Subtilis 168
BglS This gene encodes an endo-beta-1,3-1,4-glucanase (bglS), which is from the bacterium Bacillus subtilis subtilis 168. The enzyme will hydrolyze and thereby cleave internal 1,4 linkages adjacent to 1,3 linkages. Usage and Biology The substrates vulnerable to the bglS encoded enzyme are mixed linked beta-glucans. These glucans would have 1,3 and 1,4 beta linkages within the polysaccharide linking together the glucose monomers. Examples of these glucans can be found in oats, maize, and barley. (1) http://mic.sgmjournals.org/content/141/2/281.long (2) http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1984.tb04259.x/pdf
No design constraints were needed for RF10 insertion, a second stop codon was also added to the end of the coding region.
Tet Repressible N-terminal Arg Tagged EYFP (no terminator)
This is BBa_K331022 with the tetracycline repressible promoter BBa_R0040 added in front. This will be a component of a proof-of-concept part That will show localization of proteins into the interior of a microcompartment formed by the assembly of lumazine synthase proteins.
mKate-HH - Hammerhead attached behind mKate MammoBlock
analogue of BBa_I13507 (rbs+FPa, terminator) using a new fluorescent protein and terminator
The transmembrane receptor Tar of E. coli mediates chemotaxis towards aspartate.
One XbaI site was found at position 420. The restriction site was changed from TCTAGt to CCTAGt via QuikChange PCR.
The part was amplified from genomic DNA of E. coli str. K-12 substr. DH10B chromosome (NC_010473.1).
PoPS -> PhiR73 pag - PLL promoter -> PoPS
Lumazine Synthase is an enzyme which creates Lumazine, a product which aggregates forming a hollow spheroid which can act as a mirocompartment, or artificial organelle. The Lumazine forms negatively charged pores, which can be used to introduce proteins. The proteins which are being introduced into the microcompartment must be equipped with an Arginine Tag.
point mutations of EcoRI and PstI sites?
encodes enoyl CoA hydratase (cleaves CoA group from feruloyl CoA) ech converts feruloyl CoA to vanillin catalyses the final stage of the vanillin biosynthesis pathway
P. fluorescens has a high GC genome, therefore we had the coding region optimised for expression in E. coli. Gene was synthesised by geneart removed one PstI site
Pseudomonas fluorescens NCIMB 9046 Gene was synthesised by geneart Cloning and Characterization of the Ferulic Acid Catabolic Genes of Sphingomonas paucimobilis SYK-6, E Masai, et al, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 68(9): 4416???4424 (2002)
Hemeoxygenase with RBS (E. coli Codon Optimised)
E. coli codon optimised Synechocystis Heme Oxygenase with a Ribosome Binding Site (RBS), oxidises Heme into biliverdin which absorbs light in the 360 nm and 650 nm wavelength producing a green colouration.
Codon optimisation for E. coli, whilst staying within the GC constraints of gibson assembly.
This part is the mutation of Sic1, we mainly mutate the polyphosphoric acid sites of the Sic1. Here we just use it as a cell cycle regulate switch, it may let the cell cycle stop at G1 phase.
Mutate the polyphosphoric acid sites of the Sic1, at least seven sites of it.
It come from saccharomyces cerevisiae's whole genome.
pentaerythritol tetranitrate reductase with native rbs
This is the coding sequence for the enzyme pentaerythritol tetranitrate reductase (PETN reductase) of ''Enterobacter cloacae'' PB2. This enzyme reductively denitrates nitrate esters such as PETN (pentaerythritol tetranitrate, penthrite) GTN (glycerol trinitrate, nitroglycerine) and EGDN (ethylene glycol dinitrate) yielding the alcohol and nitrite ion. It can also reductively attack nitroaromatic compounds such as TNT (2,4,6-trinitrotoluene) forming initially hydride-Meisenheimer complexes which break down via an unknown route to yield nitrite and non-aromatic products. Reference: Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. (1996). Degradation of pentaerythritol tetranitrate by ''Enterobacter cloacae'' PB2. Applied and Environmental Microbiology 62(4), 1214-1219. Reference 2: French,C.E., Nicklin,S. and Bruce,N.C. 1996. Sequence and properties of pentaerythritol tetranitrate reductase from ''Enterobacter cloacae'' PB2. J. Bacteriol. 178 (22), 6623-6627 (1996).
No special considerations, but due to an error, the primers were actually designed based on the homologous gene of another strain of ''Enterobacter cloacae'', which has a very similar coding sequence but different sequence in the upstream region. The sequence shown here is the correct sequence determined by sequencing the construct.
This part was amplified from plasmid pONR1 which bears the PETN reductase gene originally cloned from ''Enterobacter cloacae'' PB2.
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)
Arsenic inducible mtrB with cut sites flanking RBS
The purpose of this part is to upregulate mtrB expression in response to arsenic. When expressed in a Shewanella strain lacking mtrB on the chromosome, this composite part will function as a component of a biosensor. Specifically, when such a complemented strain is inoculated in a microbial electrochemical system, current output will increase in response to arsenic. Within the genetic circuit, arsR acts as a negative autoregulator, repressing not only the expression of downstream mtrB, but also its own expression. Upon association with arsenic salts, ArsR dissociates from the operator of the arsenic inducible promoter, upregulating expression of downstream protein. Because mtrB activity is requisite for functionality of the mtr electron pathway in Shewanella spp., arsenic thus induces electron shuttling through the mtr pathway.
Primers were designed such that the wild-type Shine Dalgarno sequence was included upstream of the mtrB start codon, maintaining spacing. However, the appended BamHI cutsite downstream of the Shine Dalgarno sequence may alter translational efficiency.
Arsenic-sensitive component of this composite part was originally derived from the arsenic detoxification operon of E. coli strain JM109; BioBrick part BBa_J33201 was the source of DNA. mtrB is originally derived from the mtrCAB operon of Shewanella oneidensis MR-1; BioBrick part BBa_K098994 was used as DNA source.
Composite part for Biphasic switch tests.
Search for illegal sites was performed, they were found and deleted.
Du Jin,Hou Nan,Liu Duo,Zhang Weiran,Yuan Fang
HH-mKate - Hammerhead attached to front of mKate MammoBlock
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.
ALDH, an aldehyde deyhydrogenase that facilitates the third step in alkane degradation, from n-alkanals to n-alkanoic acids, which can then be further degraded through β-oxidation
This is the reconstructed gene for the EfeU iron transporter. This gene is inactive in K-12 strain E. coli, but has been characterized in the Nissle 1917 strain. (Grobe, Scherer et al. PMID: 16987175.)
The sequence was conceptually reconstructed from the frame-shifted version. The Ecogene entry has an "N" for the nucleotide which is uncertain, but a comment in that entry says that the correct amino acid is likely glycine. Thus we chose N = G in the final sequence.
From wild-type Nissle 1917 E. coli. The sequence came from the Ecogene entry EG13860.
This is a TetR mutant, who carries the V36F mutation.
The mutation was introduced by site-directed mutagenesis
The TetR gene was PCR-amplified from the Repressilator plasmid. We then introduced the V36F mutation by site-directed mutagenesis.
Lac-inducible generator of Lpp-OmpA(46-66)-Streptavidin wild-type + His6tag
This device contains a lac promoter and strong ribosome binding site for lac-inducible expression of the fusion protein of Lpp signal peptide, OmpA aa46-66, and streptavidin wild-type + His6 tag. This expression should display streptavidin on the cell surface of E. coli. NOTE ABOUT THE SEQUENCE: The mixed site between parts is 'only' six base pairs, ACTAGA. There is no spacer T or G nucleotide. These spacer nucleotides have been placed in the results for "get selected sequence" as an automatic composite-parts addition for the BioBricks mixed site between assembled parts. However, this does not apply between the R0010 and B0031 in the J04500 composite because that was assembled in a standard fashion.
There is no spacer nucleotide between the end of the device and the SpeI restriction site. Also, the mixed site in between the parts is only six base pairs long, ACTAGA, without the spacer T or G in between parts. This does not apply between R0010 and B0031.
LacI coding device regulated by pTet inverting regulator which action is inhibited by the addition of tetracycline or its analog, aTc.
This is the universal acceptor plasmid for creating new PhytoBricks parts. This sequence is contained within the BioBricks vector pSB1C3.
Inducible HipB antitoxin under the control of lac promoter
HipB is the antitoxin of the HipAB toxin-antitoxin (TA) module. HipB neutralizes the toxin effect of HipA due to protein-protein interactions.
This part was extracted of the genome of Escherichia coli K12 MG1655 using high fidelity PCR
Part to produce the appa e. coli phytase. This phytase acts to break down phytic acid into inositol, releasing chelated iron and other micronutrients.
http://www.ncbi.nlm.nih.gov/nuccore/341941295?report=fasta Genomic sequence.
This part is similar to [http://partsregistry.org/wiki/index.php/Part:BBa_I732083 BBa_I732083], but there is a mutation induced by PCR in [http://partsregistry.org/wiki/index.php/Part:BBa_I732084 BBa_I732084]. We are convinced that the mutation has nothing to do, and this part works as well as [http://partsregistry.org/wiki/index.php/Part:BBa_I732083 BBa_I732083].
PoPS -> PSP3 pag - Psid promoter -> PoPS
PoPS -> PhiR73 pag - Psid promoter -> PoPS
Fluoresceine A -binding derivative of a Lipocalin (BBP, bilin binding protein from pieris brassicae), originally designed by Arne Skerra [see Ref. 1,2].
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.
Michael Brasino, Alicia Wong, Rashmi Ravichandran
Fluoroescent(sfGFP) membrane localization (mamI)
Lpp-OmpA(46-159)-Streptavidin wild-type + His6
Assembled construct of signal peptide of lipoprotein, five transmembrane domains of outer membrane protein A, and streptavidin, wild-type with His6 tag at the end. This construct codes for a protein which displays streptavidin on the cell surface.
There are no spacer nucleotides between the part and the XbaI restriction site, or between the part and the SpeI restriction site, so that the mixed site would be 6bp long, and reading frame would be maintained between protein domains. Also, the mixed site in between the parts is only six base pairs long, ACTAGA, without the spacer T or G in between parts.