MaSp1 and MaSp2 12-mer block co-polymer under control of T7 promoter and RBS. Ratio of 1 MaSp1 to 1 MaSp2.
Human ferritins do loads more iron atoms then bacterial ferritins. Furthermore they are forming metall cores that are less contaminated with phosphates then e.g. bacterial ferritins. Due to this fact, this should result in a purer iron crystal with an improved magnetic moment.
This design was provided by iGEM Calgary and the HuFerritin as a single protein coding biobrick was constructed by iGEM Berlin as this biobrick was not accessible in this version in the registry.
iGEM Calgary 2013 from a synthesized codon optimized construct
This part consists of histidine tag and RFP. His-tag is useful for purifying RFP.
Tal Naor, Ricarina Rabinovitz, Ittai Rubinstein, Yair Maimon
The promoter Plux is induced by the binding of an AHL-luxR dimer. This bio-brick leads to the synthesis of the enzyme Green Renilla Luciferase, which catalyses a luminescent reaction. lacI can be used to repress the promoter Plac.
BBa_R0062: Plux, BBa_B0030: Strong RBS, BBa_B0030: Strong RBS, BBa_B0010:T1 (terminator) from E. coli.
This is the translational unit for the phosphoketolase (XPK). It derives from Bifidobacterium adolescentis and is codon optimized for E. coli.
The manA promoter from Xanthomonas campestris
The sequence did not have any restriction sites to remove so it was immediately PCRed out of the genome and had the registry prefix and suffix added to it. It was then ligated into the pSB1C3 vector.
The source of this part is Xanthomonas campestris pv. campestris 8004 genomic DNA which was provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
Leon Kraus, Jonathan Pletzer-Zelgert, Jonas Sindlinger, Carmen Sakas Gandullo, Sebastian Holzner, Elena Tonchevska, Mirjam Haun
Encodes for an enzyme, that is capable of cleaving A- and B-antigens in the human ABO-Bloodgroup system. As the name suggests, the galactose and connected saccharide residues are cleaved from the antigen, leaving behind the the Oh-Antigen which is characteristic of the Bombay-blood type. Part in RFC25.
Part in RFC25, in order to ease fusion with other parts, for example immobilisation tags.
The protein encoded by this sequence naturally occurs in Clostridium perfringens. This sequence was optimized for codon usage in E. coli K12 and synthesized.
quorum sensing machinery that activates GFP expression
This part is the combination of two parts, BBa_I13202 and BBa_T9002. The first part (BBa_I13202) produces a luxI enzyme under the control of a lac repressible promoter. The second part (BBa_T9002) produces luxR, which combines with the luxI to activate the promoter that controls expression of GFP.
GFP (mut3b) with SsrA-DAS+2 degradation tag
GFP (mut3b) (see part BBa_E0040) with added engineered NYADAS-ssrA degradation tag (see part BBa_M0050). The tag increases GFP turn-over rate, thus providing better temporal resolution of green fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. SsrA tags encode peptide sequence that and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The NYADAS tag encodes peptide sequence AANDENYNYDAS is reported to have low affinity to ClpX thus its mediated degradation very much depends on the concentration of SspB (ClpX mediator).[1] The two additional residues ???NY??? extends tag between SspB and ClpX binding site, thus preventing clash when both these protein are bound to tag.[3] However, be aware that exact protein degradation rate is influenced by multiple other factors: ClpXP and ClpAP protease concentrations, protein stability, Km of binding to the protease, temperature [4]. ===References=== [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] McGinness, K. E., Baker, T. a & Sauer, R. T. Engineering controllable protein degradation. Mol. Cell 22, 701???7 (2006). [4] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to GFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additional residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (DAS in this case) are recognized by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
GFP comes from part BBa_E0040, the tag sequence was obtained from part BBa_M0051.
pSB1C3-6His-F[1,2]-dCas9, segment F[1,2] fusion with dCas9, 6His tag allows for protein purification.
SsrA degradation tag as RFC[105] C-terminal insert
Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena B??scher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Sch??fer, Carolin Schmelas, Silvan Schmitz, Max Waldha
M2-6(1C3) under control of T7 promoter RBS.
RBS + NpuDnaE N-intein RFC[105] assembly construct (with His6)
Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena Buescher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Schaefer, Carolin Schmelas, Silvan Schmitz, Max Waldhauer
OmpA surface protein as RFC[105] N-terminal insert
His-tagged blc repressor fused to superfolder GFP under the control of the T7 promoter
Uses T7 promotor and a strong rbs (BBa_K525998), the blc repressor linked with superfolding GFP (sfGFP) with a His-Tag. By linking sfGFP we want to detect bound protein via fluorescence.
This is a composite part which enables a high protein expression and an easy purification of blcR fused to superfolding GFP (sfGFP). The green fluorescence of the culture is a sign for protein expression.
We clone blcR in T7-RBS-sfGFP and added a His-Tag via PCR and Gibson Assembly.
We used the biobrick (BBa_E0840) available in the database of the iGEM since 2004 to be connected to our ???essential biobrick??? (BBa_K1355001) and - together - form a system capable of biodetect mercury in bacteria. In the presence of mercury, The MerR protein will bind to it and MerT, MerP and the Green Fluorescent Protein (GFP) protein will be expressed and the biodetection will begin! The MerP and MerT protein are responsible for the transport of mercury from the periplasm to the cytoplasm and the GFP will act as rep??rter to alerting the presence of mercury in the middle and the higher concentration of mercury is, higher will be the GFP expression. And If there is no mercury in the middle, the synthesis of MerT, MerP and GFP protein will be repressed by the MerR repressor protein.
For this construct, we followed these steps: Extraction of plasmid DNA and DNA quantification of bacteria transformed with the Essential Biobrick (BBa_K1355001) and the bacteria transformed with the GFP Biobrick (BBa_E0840); Verifying the electrophoretic profile of the extracted plasmid DNA; Restriction enzyme digestion for Essential Biobrick with SpeI and PstI and for GFP Biobrick with PstI and XbaI; Checking the electrophoretic profile of digested samples; Fragment of interest purification from the gel; Ligation the gel purification of GFP gene and digested Essential Biobrick; Transformation of ligation in DH5-alpha; Plasmid DNA extraction from bacteria transformed; Check the electrophoretic profile to see results of samples linked (no fragments); Digestion of Essential Biobrick + GFP Biobrick with EcoRI and PstI, aiming to analyze the fragment size to be isolated; Checking the electrophoretic profile of the digested sample to obtain results showing that the isolated fragment is the junction of Essential Biobrick +GFP Biobrick ;
E.coli optimized green renilla luciferase under LuxR-AHL regulated promoter
To assamble this part, and others like it, we used a specialized Gibson Barcode System to allow for a modular construction of gates with various components using the same Gibson reaction conditions, allowing for the assembly of a large number of constructs in parallel to each other
GFP (mut3b) with DAS-ssrA degradation tag
GFP (mut3b) (see part BBa_E0040) with added DAS-ssrA degradation tag (see part BBa_M0052). The tag increases GFP turn-over rate, thus providing better temporal resolution of green fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. SsrA tags encode peptide sequence that is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The DAS tag encodes peptide sequence AANDENYADAS is reported to have low affinity to ClpX thus its mediated degradation very much depends on the concentration of SspB (ClpX mediator).[1] However, be aware that exact protein degradation rate is influenced by multiple other factors: ClpXP and ClpAP protease concentrations, protein stability, Km of binding to the protease, temperature [3]. ===References=== [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
GFP comes from part BBa_E0040, the tag sequence was obtained from part BBa_M0052.
Alexia Satouf, Clara Bouyx, Aimeric Agaoua, Lambert Antoni, Vincent Castel
SH3pep-LZA is a link wich mediates proteins interactions.
Amplified from pWW2181,Whitaker et al. PNAS 2012
Artilysin Art-175 fused at N-terminal with porin-dependent YebF secretion signal protein
This is the translational unit of the 6-phospho-3-hexuloisomerase (PHI) from Bacillus methanolicus MGA3. It is codon optimized for E.coli.
gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm
Bastian Wagner, Christian Mende, Andreas Schmidt, Max Dombrowsky
pFig2c: Mating factor alpha responsive promoter
pFig2c is a mating factor alpha responsive promoter, meaning that it is activated by the presence of mating factor alpha. It is endogenous in S. Cerevisiae cells and is followed by the Fig2 gene. The Fig2 gene is a part of the mating pathway in S. Cerevisiae, it contributes to maintaining cell wall integrity when the cells project ("shmoo") towards each other.
We changed nucleeotide 22 from adenine to thymine in order to eliminate the illegal Pst1 cut site.
pFig2c is found in the genomic sequence of MATa S. cerevisiae.
The part is designed to produce RFP in higher temperatures. The RNA Thermometer acts as a heat-shock RBS.
The part is composed of distinct parts from various origins.
Lead repressor under control of constitutive promoter and strong RBS
Lead promoter repressor under the control of konstitutive promoter (K608002)
J23101+B0034+VHb (Vitreoscilla haemoglobin)
Lactose/IPTG inducible promoter with RBS, RFP reporter tagged with LVA-ssrA degradation tag and terminator. The tagged RFP is actively degraded within cell, thus provides better temporal resolution of red fluorescence and promoter activity. This part can be considered as improved version of BBa_J04451 as it does not contain StuI restriction site coding for additional 'RP' residues between RFP and LVA tag.
Part was assembled using BrickClip assembly (BBF RFC 104) using other biobrick parts as templates. BrickClip assembly is a special case of more general Paperclip assembly method.[1]
Sascha Hein, Rene Sahm, Malte Blumenroth, Tobias Gabriel, Sebastian Palluk
CBDclos fused to Phytochelatin (PC) EC20
It is the combination of IAAH and termination.
We combine IAAH and the termination to improve the efficiency of the building.
RhlR regulatory protein from P. aeruginosa SQ6
Coding a transcription regulator RhlR. RhlR is necessary for transcriptional activation of the rhlAB genes encoding the rhamnosyltransferase. It also functions as a transcriptional activator of elastase structural gene (lasB). RhlR binds to autoinducer molecules BHL (N-butanoyl-L-homoserine lactone), and HHL (N-hexanoyl-L-homoserine lactone). This part includes the noncoding sequence of RhlR.
It includes both the complete coding sequence and part of noncoding sequence of RhlR.
It comes from the genomic sequence of Pseudomonas aeruginosa SQ6.
Pseudomonas AutoInducer 1 (PAI-1) Biosensor
This is a composite part that consists of 4 biobricks. This is an alternative PA1 Activated System from Bba_K1315009 that instead uses pluxR (Bba_R0062). In the front it has ptet(Bba_R0040) which is constitutively on. This produces LasR all the time with lasR(Bba_C0179) next in the sequence. Only in the presence of the Pseudomonas Autoinducer 1 (PA1), LasR forms an complex with PA1 and it can then activate the pluxR(Bba_R0062) promoter that leads to the production of GFP(Bba_E0040).
All the inserts were digested with XbaI and PstI, and all the vectors were each time cut with SpeI and PstI.
All the parts have come from existing iGEM distribution kits.
sfGFP fused to dCBD with linker in RFC 25
pSB1C3-6His-dCas9-F[1,2], this part is a fusion protein of dCas9-F[1,2], the His tag is used for protein purification
dCas9 fusion protein coding sequence is too long, so you'd better amplify it separately.
Micrococcal Nuclease (DNase) fused at N-terminal with DsbA signal sequence
The cblD promoter from Burkholderia
This is a promoter that is activated by the OmpR-like two-component response regulator BCAM0228 (Bba_K1315007) in the BCAM0227-BCAM0228 sensor system. The Burkholderia diffusible signal (BDSF) is being picked up by the BCAM0227 sensor kinase that is hypothesised to eventually lead in involvement of BCAM0228. BCAM0228 is then activating cblD.
The source of this part is Burkholderia cenocepacia J2315 genomic DNA which was provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
The BCAM0228 Response Regulator from Burkholderia
This is the coding sequence of the BCAM0228 two-component regulator of the BDSF sensory BCAM0227-BCAM0228 system. This system is found in Burkholderia spp.
The sequence had 1 EcoRI site that was mutated out by site-directed mutagenesis.
MaSp2 6 mer in pSB1C3. No regulatory elements present.
RFP from Discosoma striata (coral) with LVA-ssrA degradation tag
Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LVA-ssrA degradation tag. The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. This tag is commonly attached to repressor proteins for use in various gene networks (e.g., oscillators). The tag encodes peptide sequence AANDENYALVA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LVA tag is reported to lead to fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; protein stability; Km of binding to the protease; temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to RFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additonal residues and restriction sites. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (LVA in this case) are recognised by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
RFP comes from part BBa_E1010, tag sequence was obtained from paper by Andersen et al., (1998).[2]
S-adenosyl methionine-dependent methyltransferase relate to Met-DMSP byosynthesis pathway(SAMmt)
This part is S-adenosyl methionine-dependent methyltransferase gene of Fragilariopsis cylindrus. The gene code S-adenosyl methionine-dependent methyltransferase relate to Met-DMSP byosynthesis pathway from Fragilariopsis cylindrus. which catalyze third step of Met-DMSP biosynthesis pathway.
This part was excluded intron. And there was PstⅠ recognition site so I made single base substitution in recognition site.
GFP (mut3b) with LVA-ssrA degradation tag
GFP (mut3b) (see part BBa_E0040) with added LVA-ssrA degradation tag. The tag increases GFP turn-over rate, thus providing better temporal resolution of green fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALVA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LVA tag is reported to lead to fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations, protein stability, Km of binding to the protease, temperature [3]. References [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to GFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additonal residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (LVA in this case) are recognised by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
GFP comes from part E1010, the tag sequence was obtained from paper by Andersen et al., (1998).[2]
Luna Barroco de Lacerda, Maria Clara Tavares Astolfi
The biorremediator device is composed by our ???essential biobrick??? (BBa_K1355001) + a MerA gene (BBa_K1355000). In the presence of mercury, The MerR protein will bind to it and MerT, MerP and MerA protein will be expressed and bioremediation will start! The MerP and MerT proteins are responsible for the transport of mercury from the periplasm to the cytoplasm, leading him to the mercury ion reductase (MerA)! The beautiful MerA be responsible for the reduction from Hg2+ in Hg0, which is volatile at room temperature and able to passively leave out the bacteria.
For this construct, we followed these steps: Extraction of plasmid DNA and DNA quantification of bacteria transformed with the Essential Biobrick (BBa_K1355001) and the bacteria transformed with the MerA Biobrick (BBa_E0840); Verifying the electrophoretic profile of the extracted plasmid DNA; Restriction enzyme digestion for Essential Biobrick with SpeI and PstI and for merA Biobrick with PstI and XbaI; Checking the electrophoretic profile of digested samples; Fragment of interest purification from the gel; Ligation the gel purification of merA gene and digested Essential Biobrick; Transformation of ligation in DH5-alpha; Plasmid DNA extraction from bacteria transformed; Check the electrophoretic profile to see results of samples linked (no fragments); Digestion of Essential Biobrick + merA Biobrick with EcoRI and PstI, aiming to analyze the fragment size to be isolated; Checking the electrophoretic profile of the digested sample to obtain results showing that the isolated fragment is the junction of Essential Biobrick +merA Biobrick ;
including streptavidin, 2??linker, and firefly luciferase N terminal 416 amino acid domain.
streptavidin and N terminal 416 amino acid domain was linked by 2??linker
This is a functional coding sequence for the mercury transport proteins merT/merP from P. aeruginosa. This part will result in mercury intolerance due to greater mercury transport into engineered cells.
The Anderson promoter provides strong constitutive expression of merT/merP in E. coli
Anderson promoter is that of part BBa_J23102. Terminator is that of part BBa_B1006. merT/merP genes are naturally found in Pseudomonas aeruginosa. This part was synthesized by GenScript in the vector pUC57. The part was then transferred into pSB1C3 using standard cloning procedures.
Alexandra Goretzki, Tanja Habeck, Nina Kuschik-Maczollek, Laurin Monnheimer, Steven Pilger, Bianca Reisinger, Daniel Stumpf
Deinococcus radiodurans uracil-DNA glycosylase 2
The pqsA promoter from Pseudomonas
The Pseudomonas quinolone signal (PQS) is imperative in modulating mechanisms that enable Pseudomonas aeruginosa to survive at times of stress. P. aeruginosa is the main pathogen responsible for severe deterioration in the Cystic Fibrosis lungs. The production of this signal relies on the pqsABCDE operon. The transcriptional factor PqsR (BBa_K1315000) positively regulates this operon. http://www.ncbi.nlm.nih.gov/pubmed/25225275 http://www.ncbi.nlm.nih.gov/pubmed/16735731 http://www.ncbi.nlm.nih.gov/pubmed/21539583 http://www.ncbi.nlm.nih.gov/pubmed/?term=pqsr-dependent+and+pqsr-independent+regulation+of+motility+and+biofilm+formation+by+pqs http://www.ncbi.nlm.nih.gov/pubmed/12393198
The source of this part is the genome of Pseudomonas aeruginosa PAO1 provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
UTR-sfGFP under control of Copper responsive promoter
copper responsive promoter with 5?? untranslated region and sfGFP for detection
copper responsive promoter of ????E.coli`` with sfGFP for detection
This part is the coding sequence for the Helicobacter pylori catalase, which effectively catalyses the breakdown of hydrogen peroxide. The breakdown of hydrogen peroxide, which is an exothermic process, will be utilized to provide heat to the medium to increase temperature to counteract a shift that decreases the temperature of the host environment.
There are no specific design considerations that were made.
Helicobacter pylori XZ274 http://www.ncbi.nlm.nih.gov/protein/AFJ81452.1 GenBank: AFJ81452.1
RBS + SspGyrB C-intein RFC[105] assembly construct (with His6)
Contains an IPTG inducible promoter an RBS, RFP, and terminator.
This part is an inverting regulator sensitive to LacI (BBa_C0010, BBa_C0012, etc.) The T7-lac promoter is a hybrid regulatory region consisting of the T7 promoter of T7 phage next to LacO. The hybrid design allows for strong promotion that can nevertheless be:  repressed by LacI  induced by lac or IPTG, showing a high expression level.  CAP protein will not influence the expression. Strong promoter. Only E.Coli BL21(DE3) can use this part.
This part contains all features from [http://parts.igem.org/Part:BBa_K1758102 BBa_K1758102] plus the blc-operator site described in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083662/ Pan et al. 2013]. BlcR (see http://parts.igem.org/Part:BBa_K1758370 K1758370]) can bind to the operator site.
This part was assembled via PCR with Gibson primers on [http://parts.igem.org/Part:BBa_K1758102 BBa_K1758102]
NisA codes for the nisin precursor. Together with the genes NisB, NisT, NisC, NisP, NisR and NisK it is responsible for producing the lantibiotic nisin in Lactococcus lactis. Can be used in the Lactococcus lactis NZ9800, a strain with a deletion in the original NisA gene, to let it produce nisin.
Obtained by PCR on the genome of L. lactis NZ9700, a nisin producing strain.
Christian Sator,Stefan Zens, Max Zander, Benedikt Spannenkrebs, Sebastian Jaeger
The product of the RraA-gene has been reported to interact with RNase E from ''E. coli'' and to alternate its activity. Airen showed that if RraA-protein is added to a cell free protein synthesis reaction, the productivity raises about 30% (Airen, 2011). He postulated that activity of RNase E in the reaction is lowered due to the interaction with RraA-protein, therefore mRNA-levels are stabilized. RNase E is brought to the ''in vitro'' reaction by ''E. coli'' cell extract itself.
'''Amplification with Gibson-primer:''' *Amplification of regulator of ribonuclease activity A (''RraA''): '''Forward primer RraA''' 5'-ATGAAATACGATACTTCCGAGCTTTG-3' '''Reverse primer RraA''' 5'-TCATTCAATATCCAGCGGATCTTC-3' *Amplification of the pSB1C3-Backbone: '''Forward primer pSB1C3 RraA(43 bp)''' 5'-TCAGAAGATCCGCTGGATATTGAATGATACTAGTAGCGGCCGCTGCAG-3' '''Reverse primer pSB1C3 RraA(43 bp)''' 5'-TTCTTCCAGTTGTTCCGGGTTTGCCATCTCTAGAAGCGGCCGCGAATTCC-3' '''Ligation via [http://2015.igem.org/Team:Bielefeld-CeBiTec/Protocols Gibson-Assembly].'''
RraA was obtained via PCR on ''E. coli'' K12 genomic DNA.
Aldose reductase (GRE3) from Saccharomyces cerevisiae S288c
Alexandra Goretzki, Tanja Habeck, Nina Kuschik-Maczollek, Laurin Monnheimer, Steven Pilger, Bianca Reisinger, Daniel Stumpf, Sebastian Barthel
Escherichia coli CRP protein is a cAMP-activated global transcription factor. CRP protein binds cAMP and leads to the activation of transcription of more than one hundred promoters. This protein enhances the possibility of RNA polymerase holoenzyme binding and initiating transcription.
Introduced a mutation from ACTTCA to ATAATT.
GFP (mut3b) with LAA-ssrA degradation tag
GFP (mut3b) (see part BBa_E0040) with added LAA-ssrA degradation tag (see part BBa_M0050). The tag increases GFP turn-over rate, thus providing better temporal resolution of green fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes E.coli wild type ssrA peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is reported to lead to fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations, protein stability, Km of binding to the protease, temperature [3]. ===References=== [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to GFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additional residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (LAA in this case) are recognized by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
GFP comes from part BBa_E1010, the tag sequence was obtained from part BBa_M0050.
Sebastian Barthel, Niklas Hummel, Jasmin Weber, Michelle Zoeller, Marcus Geissler, Stefan Bohn, Simon Linder
Chromium responsive promoter with UTR+RBS and sfGFP
Chromium responsive promoter with 5?? untremslated region and sfGFP for detection
Apis mellifera (honeybee) silk fibroin 3 with regulatory elements
Same as part BBa_K1763000 with regulatory elements upstream. These include lac promoter and rbs (will update later)
Apis mellifera genome. Reference Genbank FJ235090.1
Inducible generator of Humicola insolens cutinase (HIC) with N-terminal His-Tag
Sebastian Barthel, Steven Pilger, Jonas Sindlinger, Bianca Reisinger, Sven Jager
This construct encodes a truncated version of E. coli W3110 SerA ( D-3-phosphoglycerate dehydrogenase), missing the last 75 aa of the full sequence, and without stop codon. SerA is requiered for serine biosynthesis. Following the work of Peters-Wendisch et al. 2005, this mutated version of the enzyme is expected to be no-longer inhibited by Serine, and to allow the production of a high serine concentration.
The part was obtained by PCR from E.coli strain W3110 and SLIC assembly. The construction removed the EcoR1 restriction sites in the gene by the silent mutations of a GAA codon to a GAG codon.
Amplified by PCR from E.coli W3110 genomic DNA.
Response regulator of the cusCFBA-cusRS divergent operons fused with a leucine zipper.
Amplified from pWW2021,Whitaker et al. PNAS 2012
The M13ori-pCas9 composite part consists of the M13 origin of replication (M13ori) and the pCas9 part (BBa_K1218011) containing a tracrRNA sequence, the cas9 gene, and a minimal CRISPR array. M13ori (BBa_K1445000) is a 500bp noncoding sequence that is recognized by the gene II of the M13 phage, which results in the uptake of the plasmid containing the ori into an assembled phage coat. This part does not contain any complete phage genes so phage production requires a Helper Phagemid such as M13K07. There will be some packaging of Helper Phagemid but their packaging signal weakened so parts containing the M13ori are preferentially packaged. pCas9 contains the native trcrRNA and promoter region upstream of the Cas9 protein. The type II Cas9 protein is the native, active version from streptococcus pyogenes with the exceptrion of a SNP to erase an illegal EcoRI cleavage site. Downstream of the Cas9 protein is a minimal CRISPR array. This minimal array only includes two CRISPR repeats flanking a spacer region. It is this spacer region that determines which DNA sequence is targeted by the Cas9 endonuclease. Spacer sequence can be easily replaced with the protocol found here.
We have experienced some toxic effects by the pCas9 part so we suggest growing part-containing cells in lower concentrations of antibiotic, even if plasmid contains a high copy origin of replication.
The M13 origin of replication from Litmus28i and part BBa_K1218011 from the registry.
The aresnic inducible banana odor generator converts isoamyl alcohol to isoamyl acetate in the presence of arsenic.
We digested the Biobricks with CIP to assure that the plasmids did not re-ligate to themselves.
The arsenic promoter with the arsR repressor gene is naturally found in E. coli. The ribosome binding site, the ATF1 gene, and the double terminator combined sequence was obtained from the Biobrick BBa_J45199.
RBS+NpuDnaE C-intein RFC[105] assembly construct (with His6)
This intein assembly construct is part of the iGEM team Heidelberg 2014's strategy for cloning with split inteins. Inteins are naturally occuring peptide sequences that splice out of a precursor protein and attach the remaining ends together to form a new protein. When splitting those intein sequence into an N-terminal and a C-terminal split intein one is left with a powerful tool to post-translationally modify whole proteins on the amino-acid sequence level. This construct was designed to express any protein of interest fused to the Nostoc punctiforme DnaE N-terminal split intein. The corresponding C-terminal construct is BBa_K1362101 . Upon coexpression or mixture of the N- and C-constructs protein splicing takes place and the N- and C-terminal proteins of interest are irreversibly assembled via a newly formed peptide bond. This mechanism can be applied for a variety of different uses such as the activation of a protein through reconstitution of individually expressed split halves. See our [http://2014.igem.org/Team:Heidelberg split sfGFP experiment] and the respective parts in the registry for more information. Protein splicing offers many new possibilities and we hope to have set a foundation that you guys can build on! ===Nostoc punctiforme DnaE split intein (NpuDnaE)=== Inteins can be found in all kingdoms of life, however their use for the organism remains yet unknown. In the past years many intein sequences were found in the genomes of cyanobacteria. NpuDnaE is a naturally split intein from the alpha subunit of Dna Polymerase III in Nostoc punctiforme bacteria which shows [[#References|[2]]] extraordinarly high splicing activity and therefore is on of the gold-standards for inteins since many years. Amino Acid Sequence: N-Intein: AEY | CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN C-Intein: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN | CFN
The part is assembled from different sources. The intein sequences come addgene or were kindly provided by different working groups. BBa_J04450 was used as mRFP selection marker. The scars and overhangs are part of the RFC[???] intein cloning strategy and are further explained on our [http://2014.igem.org/Team:Heidelberg/parts/rfc wiki] and in RFC[???] [[#Reference|[1]]]. Please see the corresponding basic parts for the individual detailed information.
Laura Helleckes, Michael Osthege, Sarah Lubjuhn
This is the translational unit of GlgA (Glycogen synthase) for elongation of glycogen chains. Through autophosphorylation, GlgA is also the starting point of glycogen synthesis in bacteria.
amilCP blue chromoprotein with RBS and promoter
This plasmid contains the amilCP blue chromoprotein gene found in part BBa_K592009 and the RBS and promoter from part BBa_K608002.
This part is made from parts BBa_K608002 and BBa_K592009.
pRha - fimH 136 KO - RPMrel - SpyTag_225 - HisTag_225
mRFP generator under control of T7 promoter
mRFP is synthesized when T7-polymerase is induced.
Both subparts were obtained from iGEM distribution plates.
This part is the Amino Half of GFP with termination codon
Because of the need of our project, we added a termination codon when designing the PCR.
This part is gained from part BBa_I715019. we added the termination codon when designing the PCR.
RBS + TerDnaE3 C-intein RFC[105] assembly construct (with His6)
This part is the full renilla luciferase. The split protein has also been submitted as biobricks: BBa_K1486021.
Jonas Sindlinger, Leon Kraus, Jonathan Pletzer-Zelgert, Elena Tonchevska, Sebastian Holzner, Mirjam Haun, Sebastian Barthel
RBS + GST (glutathione-S-transferase)-CRS5 (metallothionein) + Ter
his part serves as a functional metallothionein for heavy metal binding and conferred resistance to heavy metals when combined with an appropriate promoter. CRS5 gene is the metallothionein that binds to heavy metals and it is fused to GST for stability.
The part contains a KpnI cut site between the biobrick prefix and the RBS that is a result of removing the T7 promoter from part BBa_K1460001.
This part is BBa_K1460001 with the T7 promoter removed.
Peptide 1018 is a synthetic cationic peptides, derived from natural peptides such as the human cathelicidin LL-37 and the bovine peptide indolicidin. This part work as biofilm inhibitory compounds. Anti-biofilm peptides are similar to cationic antimicrobial peptides (which are active against planktonic bacteria), comprising both cationic and hydrophobic amino acids.
This part will have broad spectrum effect on gram-negative and gram-positive bacterias
RBS + SspGyrB N-intein RFC[105] assembly construct (with His6)
In an effort to show proof of concept for the CBDA synthase portion of the project, the GSU-iGem team is using horseradish peroxidase transformed into agrobacterium then producing transgenic tobacco plants.
It is intended to be put into pGAP vectors and pORE vectors for expression in Pichia Pastoris and Agrobacterium.
Encodes for an enzyme, that is capable of cleaving off N-Acetlygalactosamine from A-group blood antigens, thereby turning them into H-antigens. Can be used to convert erythrocytes from blood type A to O. Part in RFC25 standard.
The protein encoded by this sequence naturally occurs in Elizabethkingia meningoseptica( also called Chryseobacterium meningosepticum or Flavobacterium meningosepticum). This sequence was optimized for codon usage in E. coli K12 and synthesized.
His-tagged arsenic repressor (ArsR) under control of the T7 promoter
This part can be used for expression and purification of the arsenic repressor ArsR.
HisTag-GFP_Si4-Tag fusion protein with RBS BBa_B0034
Jasmin Weber, Michelle Zoeller, Marcus Geissler, Stefan Bohn, Simon Linder, Sebastian Barthel
P170 promoter is upregulated at low pH during the transition to stationary phase.P170 was induced between pH 6.5 and pH 6.0.
P170 segment contains a cis-acting sequence involved in the control of promoter regulation.
MaSp1 and MaSp2 12-mer block co-polymer in pSB1C3, no regulatory elements. Ratio of 2 MaSp1 to 1 MaSp2.
This is the translational unit of 3-hexulose-6-phosphate synthase (HPS) from Bacillus methanolicus MGA3. It is codon optimized for E.coli.
BBa_IA Itaconic acid is an organic, dicarboxylic acid that is biotechnologically synthesized in Aspergillus terreus. It is derived from citric acid via 2 intermediates and a final decarboxylation. To enable this pathway in Escherichia coli it is necessary to introduce 3 genes. 2 of which are already established in the citric acid cycle and one that is from Apergillus terreus, a cis-aconitate decarboxylase (cadA). Figure 1 Reaction scheme of the itaconic acid producing operon. The substrates for the reaction are acetyl-coa and oxaloacetate. To simplify the process we are only looking at oxaloacetate right now. Oxaloacetate is metabolized to itaconic acid in 3 steps. ===Usage=== This part is a composite of three genes, each with a strong RBS (BBa_B0034) and all of them under control of one T7 Promoter (BBa_K1497017). citrate synthase - gltA (BBa_K1033001) aconitate hydratase 1 - acnA (BBa_K1033000) cis-aconitate decarboxylase - cadA (BBa_K1497100) iGEM TU Darmstadt 2014 :) Figure 2 Genetic map of the itaconic acid producing operon with T7 promoter. This brick directs the flux towards and finally enables E.Coli BL21 cells to synthesize itaconic acid in presence of the inductor IPTG.
Artilysin Art-175 fused at N-terminal with flagellin 26-47 peptide segment
Artilysins are an exciting class of enzyme-based antibacterials. Their name is derived from "artificial endolysin" and they exploit the lytic power of bacteriophage-encoded endolyins. Endolysins are peptidoglycan hydrolases produced at the end of the lytic cycle that pass through the cytoplasmic membrane, degrade the peptidoglycan layer and cause the osmotic lysis of the infected bacterial cell, thus liberating the progeny. Purified endolysins have been used to kill Gram-positive pathogens. Gram-negative bacteria, however, have a protective outer membrane containing lipopolysaccharide (LPS) that serves as a barrier against the peptidoglycan hydolytic activity of endolysins from the outside. To overcome this problem, selected polycationic or amphipathic peptides that locally destabilize the LPS layer have been covalently fused to endolysins. Biers et al. fused the sheep myeloid antimicrobial peptide (SMAP-29) to the N terminus of the endolysin KZ144 to create Artilysin 175. Art-175 has been shown to be a highly potent antibacterial that acts in minutes to kill virtually all Pseudomonas aeruginosa strains. [1] BBa_K1659001 is a composite of Art-175 with the 26-47 segment of Salmonella typhimurium flagellin combined with a hexahistidine tag. [2] Fla 26-47 is capable of mediating flagellar export of recombinant proteins and, combined with a hexahistidine tag, facilitates their secreted expression and easy purification. [1] Briers, Y., Walmagh, M., Grymonprez, B., Biebl, M., Pirnay, J. P., Defraine, V., ??? Lavigne, R. (2014). Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 58(7), 3774???3784. http://doi.org/10.1128/AAC.02668-14 [2] Balb??s, P., & Lorence, A. (2004). Recombinant Gene Expression. Reviews and Protocols, 267, 506. http://doi.org/10.1007/978-1-61779-433-9
Export signal combined with hexahistidine tag
Art-175 amino acid sequence: Briers, Y., Walmagh, M., Grymonprez, B., Biebl, M., Pirnay, J. P., Defraine, V., ??? Lavigne, R. (2014). Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 58(7), 3774???3784. http://doi.org/10.1128/AAC.02668-14 Amino acid sequence of Fla 26-47: Balb??s, P., & Lorence, A. (2004). Recombinant Gene Expression. Reviews and Protocols, 267, 506. http://doi.org/10.1007/978-1-61779-433-9
GoldenGate compatible AmilCP, Blue chromoprotein
A variation of the part BBa_K592009 that has been made compatible with the GoldenGate MoClo Assembly Standard as it is free from BsaI and BpiI recognition sequences. The chromoprotein naturally exhibits strong blue color when expressed. Codon-optimised for expression in dicotyledenous plants.
Domesticated for GoldenGate by removal of internal BsaI and BpiI recognition sequences. Codon-optimised for expression in dicotyledenous plants.
CBDcenA with linker fused to NiBP driven by LacI
Neomycin Phosphotransferase, for a composite biobrick.
UbiX is a flavin prenyltransferase required in ubiquinone synthesis in Escherichia coli.
SmtA metallothionein fused to linker-dCBD
We choose a strong promoter+ strong RBS to construct L-Lactate part. The NAD+-dependent L-lactate dehydrogenase gene is from Lactobacillus casei.
Coding region for cellulose binding domain. When fused to a protein, protein attaches to cellulose
Sequence was optimized for Escherichia coli and Arabidopsis thaliana codon usage.
Domain derived from cellulose binding protein from Chlostridium cellulovorans(WP_013291799).
MaSp1 and MaSp2 12-mer block co-polymer in pSB1C3. No regulatory elements present. Ratio of 1 MaSp1 to 1 MaSp2.
gRNA for SaCas9 targeting human ubiquitin B gene under U6 promoter
gRNA for SaCas9 targeting ubiquitin B under U6 promoter
NiBP fused to dCBD with linker in RFC 25
Dispersin B fused with DsbA, with mutation at nucleotide 636 (T to C)
zwf - Glucose-6-Phosphate-Dehydrogenase from E.coli
mRFP under control of the chromium responsive promoter
chromium responsive Promoter with RFP for detecition
chromium VI responsive promoter of ????Ochrobactrum tritici???? 5bvl1
The rpfG gene from Xanthomonas campestris
This is the coding sequence for the RpfG(regulation of pathogenicity factor G) protein. It is the regulator in the two-component signal transduction system of Xanthomonas campestris pv. campestris that is activated by the sensor kinase RpfC(Bba_K1315002) in the presence of DSF(diffusible signal factor).
The source of this part is Xanthomonas campestris pv. campestris(Xcc) genomic DNA which was provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
Inclusion of this part on a plasmid will facilitate the uptake of the plasmid as a single-stranded phagemid into the M13 phage. Note that this part does not code for phage coat proteins so phage containing this M13 ori and your part will not multiply. Therefore, the M13K07 or similar helper phagemid is required for phage production. This part is packaged preferentially over M13K07, reducing the risk of uncontrolled phage multiplication.
Orientation in relation to ori may affect packaging ability. Also, 3??? end contains the promoter for the M13 gene II. This is a strong promoter and may cause unwanted expression of down stream genes. We recommend placing coding regions upstream of part.
Secretion signal peptide originated from the B.subtilis extracellular protease AprE
fim switch[default OFF](Tokyo_Tech/J23119)
Sortase A circularization construct (with Smt3 and His6)
MaSp1 9 mer in pSB1C3, no regulatory elements present.
Pblc is the promoter of the blcABC operon from ''Agrobacterium tumefaciens''. It contains operator sites that are recognized by the repressor [http://parts.igem.org/Part:BBa_K1758370 BlcR]. Pblc was described by [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083662/ Pan et al. 2013].
Pblc was described by [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083662/ Pan et al. 2013].
Pblc was cloned in front of [http://parts.igem.org/Part:BBa_K516030 BBa_K516030] in pSB1C3 with Gibson primers.
This part contains the core sequence of MaSp2 that has been assembled with a 5'-AGTT-3' overhang on the 5' end of the sequence and 3'-ACAG-5' overhang on the 3' end of the sequence. This part was designed for use with ICA, along with parts BBa_K1763003 and BBa_K1763004 as an efficient method to assemble multiple MaSp2 sequences together. This setup is to show that this particular set of sticky ends is suitable for ICA. Before using in ICA, this part should be digested with BsaI, a type IIs restriction enzyme, which cuts DNA outside of its recognition site. Sequence and Features
The gene sequence for MaSp2 is particularly GC-rich, which may complicate PCR.
This part is derived from the MaSp2 gene found in the spider N. Clavipes.
Copper responsive promoter with T7-promoter and UTR-sfGFP
The glmS riboswitch has a self-cleavage activity through which it regulates gene expression.
including firefly luciferase C terminal 398 amino acid domain, 2??linker, and streptavidin.
firefly luciferase C terminal 398 amino acid domain and streptavidin are linked by 2??linker
the coding sequence of the fusion protein of dCas and the CFluc of the luciferase
Major Spidroin Protein 1 (MaSp1) with Sticky Ends CA
This part contains the core sequence of MaSp1 that has been assembled with a 5'-AGTT-3' overhang on the 5' end of the sequence and 3'-ACAG-5' overhang on the 3' end of the sequence. This part was designed for use with ICA, along with parts BBa_K1763003 and BBa_K1763004 as an efficient method to assemble multiple MaSp sequences together. This setup is to show that this particular set of sticky ends is suitable for ICA. Before using in ICA, this part should be digested with BsaI, a type IIs restriction enzyme, which cuts DNA outside of its recognition site.
RBS + SspDnaB N-intein RFC[105] non-splicing control construct (with His6)
This is a composite part that consists of 4 biobricks. In the front it has ptet(Bba_R0040) which is constitutively on. This produces LasR all the time with lasR(Bba_C0179) next in the sequence. Only in the presence of the Pseudomonas Autoinducer 1 (PA1), LasR forms an complex with PA1 and it can then activate the plasB(Bba_R0079) promoter that leads to the production of GFP(Bba_E0040).
MDFA is an antiporter which can make E-coli have a ability to tolerate high alkiline environment up to pH 10.
We use pcr to get the target genes from E-coli and invert them into pet28a which can multicopy.
MaSp1 9 mer under control of T7 promoter and RBS
Pseudomonas AutoInducer-1 response unit (lasB promoter)
The kanamycin resistance gene tn903 aph from e.coli for use with Synechocystis sp. PCC 6803. The kanamycin gene to allow transformation in Synechocystis sp. PCC 6803 (Poteete et al, 2006).
genomic DNA of kanamycin containing strain of Synechocystis sp. PCC 6803
Mytilus californianus foot protein 3(Mcfp3) variant 3
This is the cell display system of Yarrowia lipolytica, composed of LIP prepro, interest protein, and YLcwp3. LIP prepro is signal peptide used to secrete the interest protein out of the cell, and the YLcwp3 is the anchor domain binding the interest protein to the cell wall of yeast. We use this system to display silica-tag and test its binding characteristics. E.coli ribosomal protein L2 was found to bind tightly to silicon particles, which have surfaces that are oxidized to silica. This L2 silica-binding tag, called the ??????Si-tag,?????? can be used for one-step targeting of functional proteins on silica surfaces. The silica-binding domains of E. coli L2 was mapped to amino acids 1???60, 61-202 and 203???273. We respectively test the silica-binding characteristics of this three regions and their combinations. E. coli ribosomal protein L2 (1-60) is the amino acids 1???60 of E. coli ribosomal protein L2, we call it si-tag1.
We got the LIP prepro and YLcwp3 sequence from our lab, and synthetize the E. coli ribosomal protein L2 (1-60) by IDT.
The gene encoding trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA) was isolated from Pseudomonas fluorescens N3, an environmental strain able to degrade naphthalene.
Pseudomonas fluorescens (strain: PC20, biotype: F) NCBI Gene ID: 7871527
Smt3-Murine DNMT1(731-1602) fusion protein (with His6)
Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena Büscher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Schäfer, Carolin Schmelas, Silvan Schmitz, Max Waldha
* Smt3: ''Saccharomyces cerevisiae'' * DNMT1: ''Mus musculus''
Nickel Binding Protein (NiBP) Freiburg format (RFC[25])
Nickel Binding Protein in Freiburg format (RFC 25) to allow for easy use in fusion proteins.
OEPCR primers were designed to allow for the replacement of the standard BioBrick prefix and suffix with the Freiburg prefix and suffix.
This part is based on the existing BioBrick BBa_K1151001 by the 2013 Salente Lecce iGEM team.
Codes for the sfGFP protein, an optimized fluorescent reporter. Was originally optimized for Bacillus subtilis, but then showed high fluorescence in Lactococcus lactis.
Used primers: forward - 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGATGTCAAAAGGAGAAGAGCT-3' reverse - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTACTTATAAAGCTCAT-3' to PCR sfGFP(Bs) from the plasmid and add the BioBrick prefix and suffix.
PCR on plasmid MG_sfgfp(Bs), used in the study 'Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging', see references. Plasmid kindly provided by the Molecular Genetics group of the Rijksuniversiteit Groningen.
Sortase A circularization construct (with His6; without RBS)
Encodes for an enzyme, that is capable of cleaving off galactose from B-group blood antigens, thereby turning them into H-antigens. Can be used to convert erythrocytes from blood type A to O. Part in RFC25 standard.
The protein encoded by this sequence naturally occurs in Bacteroides fragilis. This sequence was optimized for codon usage in E. coli K12 and synthesized.
This part is the coding sequence of tryptophan-2-mono-oxygenase. This enzyme catalyzes the oxidative carboxylation of L-tryptophan to indole-3-acetamide which acts as an intermediate to the production of indole-3-acetic acid in the IAM pathway.
The reason why er designed this part is that we needed IAA producing pathway as a reporter in our system, and the IAAM and IAAH CDS needed to be fused with the coding sequence of the TAL effector we engineered. So we used the part BBa_K515100, which contains the CDS of IaaM and IaaH, and used PCR to eliminate the RBS sequence from the 5' region of the CDS.
This part is gained from BBa_K515100 by PCR. The IAA producing pathway originates from Pseudomonas savastanoi.
RBS + TerDnaE3 N-intein RFC[105] assembly construct (with His6)
Deinococcus radiodurans uracil-DNA glycosylase 1
RBS + SspDnaX N-intein RFC[105] assembly construct (with His6)
To test the RdRP against a control, we used a deliberately mutated version of itself. To mutate it, we used the restriction enzyme BgLII (code: agatct). This was not thought through but arbitrarily chosen, and resulted in a shift of frame and the introduction of a stop codon (tga).
Principally, our goal with this part was to create a non-functioning version of our RdRP which we could test it against, to ensure it was the RdRP proper that was having the desired effect of replicating our RNA.
The source from which this mutant is derived is our RdRP proper, part BBa_K1442100.
ranscriptionfactor，the ipgC???mxiE complex activates transcriptionfrom the pipaH* promoter
gene clusters encoding type III secretion systems
Lactose/IPTG inducible promoter with RBS, RFP reporter tagged with LAA-ssrA degradation tag and terminator. The tagged RFP is actively degraded within cell, thus provides better temporal resolution of red fluorescence and promoter activity.
Bacterioferritin (BFR) M52H heme-deletion
The M52H Mutagensis leads to an deletion of the BFR heme residue. As a result, BFR will have a reduced iron release rate and keep it's iron content inside of the protein scaffold.
CBDcenA with linker fused to NiBP in Freiburg format (RFC 25)
SYFP2 is synthesized when T7-polymerase is induced.
3A assembly of [http://parts.igem.org/Part:BBa_K525998 BBa_K525998] and [http://parts.igem.org/Part:BBa_K864100 BBa_K864100]
Biosensor device for detection of GHB and GBL
This part serves as a generator of sfGFP when induced.
Translation enhancing 5-UTR + mRFP under control of T7 promoter
mRFP generator ([http://parts.igem.org/Part:BBa_K516030 BBa_K516030]) under control of the T7 promoter ([http://parts.igem.org/Part:BBa_I719005 BBa_I719005]). The part includes a translation enhancing sequence, 5'-UTR, which improves the binding of the mRNA to the ribosome ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]). The 5'-UTR sequence contains a spacer of 10 adenine bases which has been shown to further enhance translation efficiency ([http://www.ncbi.nlm.nih.gov/pubmed/23927491 Takahashi et al. 2013]).
See [http://parts.igem.org/Part:BBa_K1758102:Design BBa_K1758102]
This part was obtained via Gibson primers that bind on [http://parts.igem.org/Part:BBa_K1758105 BBa_K1758105].
lead responsive promoter with mRFP for detection
lead responsive promoter of ????Cupriavidus metallidurans???? CH34
Unit for RFC 110 assembly, clonable in RFC 10 vectors like pSB1C3.
Standard assembly construct for RFC 110 assembly
Sortase A non-circularization construct (with Smt3 and His6)
mRFP1 controlled by the GABA-inducible activator GabR
gabR encodes a repressor which controls the expression of mRFP1.
This device was created by 3A assembly and subsequent subcloning into pSB1C3.
BBa_K1758350 was obtained by gene synthesis. BBa_K516030 was part of the distribution.
L-Lactate producing module from Lactobacillus casei
The ldh gene can produce L-lactate from pyruvate, which not only increase the overall amounts of lactate but reduce the byproduct generation.
Michael Osthege, Sarah Lubjuhn, Laura Helleckes
This is the translational unit of GlgC. It creates ADP-glucose from glucose-1-phosphate for glycogen synthesis.
Ferulic acid decarboxylase (FDC) with T7 promoter and RBS
Site directed mutagenesis of SpeI restriction enzyme site within sequence.
PelB leading sequence as RFC[105] N-terminal insert
This part is a promoter. It has a strong RBS restriction site following it which can guarantee the high efficiency of our project.
We want to construct a promoter which has RBS30 itself for RBS30 is small. If we supply a promoter with RBS, the efficiency of building will be high.
eYFP with C-terminal SH3 ligand and RBS BBa_B0034
The pre-region of lipase 2 from Yarrowia lipolytica corresponds to the signal sequence; the dipeptides XA and XP are substrates for diamino-peptidase; the dibasic KR cleavage site is substrate for Xpr6p endoproteinase.
cell division topological specificity factor MinE
Split sfGFP reconstitution positive control
Split sfGFP reconstitution N-terminal splicing half
we REMOVED the extra base from part BBa_I715020(easier to fuse with other proteins) when designing PCR primers to make the protein fusion easier.
This part is gained from part BBa_I715020.
Dispersin B with mutation at nucleotide 582 (T to C)
Monomeric Red Fluorescent Protein from Discosoma striata
Mutant of the Biobrick BBa_E1010, in order to make it compatible with the Freiburg-Standard, two AgeI-Restriction sites were deleted monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm
The Biobrick has the Following Prefix and Suffix: Gatcgaattccgcggccgcttctagataaggaggagccggc Gatcctgcagcggccgctactagtattaaccggt
NFluc of lactamase fused with dCas9, His tag allows for protein purification
Nlact for N terminal of β-Lactamase
Honeybee silk protein driven by T7 promoter
This part is the Apis Mellifera (Honeybee) silk protein coding region, with a t7 promoter element upstream as well as a ribosomal binding site.
Keep in mind that in order for the promoter to work as expected, the plasmid must be transformed into a cell line that contains a gene coding for T7 polymerase.
Genbank FJ235090.1 Genomic sequence from Apis Mellifera
sfGFP in Freiburg format (RFC 25) to allow for easy use in fusion proteins.
RBS + SspDnaB C-intein RFC[105] assembly construct (with His6)
Studies investigating the potential for co-spinning, a process in which the terminal domains of the functional B. mori silk protein heavy chain are fused with genes encoding functional binding domains, and subsequently are combined with native silk proteins to form crystalline fibers, have shown some success (Sponner et al., 2005). In vivo co-spinning simulations have previously been conducted using the transgene in silkworms, producing functionally fluorescent N-terminal ??? GFP- C-terminal fusions that successfully incorporate into endogenous Bombyx mori silkworm silk monomers (Kojima et. al., 2007). These N-Terminal and C-terminal regions are not only easier to clone due to their non-repetitive structures, but additionally allows for storage and assembly of endogenous ampullate spidroins (Askarieh et al., 2010). Composite silkworm-spider silk chimeras have shown improved mechanical properties, and may increase the ease in high-yield production due to the easier manipulation of silkworm silk monomers (Teule, et. al. 2012). Yet, this process still relies on the use of transgenic silkworms, which have longer developmental cycles, require larger storage and nutrient conditions, and difficulty to harvest relative to bacterial counterparts (Murphy et al., 2009). An ex situ co-spinning process has been developed for the production of composite N. clavipes ??? B. mori silk fibers with functional protein fusions in bacterial chassis (namely, E. coli). This process results in the construction of a transgene that can be modularized using restriction enzymes to incorporate a wide range of functional peptide binding sites or other proteins, including avidin-biotin binding domains, RGD-cadherin motifs, albumin binding domains, or antibody affinity domains (Jansson et al., 2014). Specifically, this composite structure focuses on the ability to express functional sfGFP in silk fibers as a marker for successful co-spinning, and verifies proper NC-sfGFP expression using SDS-PAGE analysis. A Standard Registry part encoding sfGFP (BBa_K515005) flanked by conserved B. mori N- and C- silk fibroin heavy chain terminal domains (NC-sfGFP) has been produced. This part is ligated in the standard registry pSB1C3 vector backbone, and transformed into electrocompetent Escherichia coli cell cultures. Expression of the fusion protein is induced using IPTG, and extracts are purified using Immobilized Metal Affinity Chromatography (IMAC) using a Ni-NTA Sepharose resin bed designed to pull down polyhistidine tagged protein constructs.
We decided to use a LacI promoter to regulate expression of the large protein construct in E. coli K12 laboratory strains, in lieu of utilizing a T7 promoter or constitutive Anderson family promoters. This is to maximize the protein yield of the co-spinning module without causing a tradeoff between regular cell growth and housekeeping protein synthesis. Additionally, we decided to use sfGFP as the reporter gene, instead of a standard mut3b GFP, due to the ability of sfGFP to function even when fused to large bulky protein domains (in this case, FibNT and FibCT). Lastly, we designed BsaI TypeIIS restriction sites flanking the reporter gene that when cut, generates sticky ends compatible with EcoRI and SpeI sites. This was created to allow iGEM teams to take protein domains of interest from the Standard Registry of Parts, and fuse them into the co-spinning module by a simple Digest and Ligate assembly to swap the sfGFP construct. However, the protein domain to be added must have the Silver Fusion prefix and suffix, to allow for in-frame cloning of the protein domain. Without this in-frame cloning, the scar sites of the tradition prefix and suffix will cause a frameshift mutation that mutate the translation product, and renders it non-functional.
The LacI promoter (BBa_R0010), RBS (BBa_B0034), and super folder GFP (BBa_I746916) were derived from sequences found in the Standard Registry of Parts. The Bombyx mori silk fibroin heavy chain N-terminal (FibNT) and C-terminal (FibCT) domains were derived from the sequenced consensus genome of Bombyx mori silkworm species, and were synthesized using IDT gBlocks gene fragments.
BsaI Restriction Site (EcoRI Compatible)
Nikolas Layer, Philipp H. O. Mayer and Philip Roessler
N-Intein part of the ssp GyrB split intein. Can be used in conjunction with the 6 amino acid long C-Intein to N-terminally immobilize a peptide onto a matrix. For immobilisation, parts need to be fused to the C-terminus of the N-intein. The N-terminus of the C-Intein is fused the matrix. The split intein recombines and splices itself out, thereby specificially immobilising the N-terminus of the peptide to the matrix. The sequence of the corresponding C-Intein is: H2N-GVPVHN-COOH (was aquired by peptide synthesis and is thus not part of the registry) This part can also be used in order to create fusion proteins, by attaching the C-Intein to a proein instead of the matrix. Part in RFC25 standard.
Part was designed in RFC25, since it is specifically used for fusion of peptides
Intein of DNA grase subunit B of the cyanobacterium Synechocystis species, strain PCC6804. Sequence optimized for codon usage in E. coli K12 and synthesised.
araC-pBAD + β-carotene + J23100 + blh
This biobrick is obtained by the assembly of two parts K1604020 and K1604021. The composite part contains the five genes necessary for the retinal synthesis. The first part contains ctrEIBY under araC-pBAD, these four gene are responsive of beta-carotene production starting from FPP, a colurless precurse presents in E.coli. The second part contains blh under costitutive promoter. It encodes for the beta-carotene15-15'dioxygenase that catalizes the cleavege of a single molecule of beta-carotene into two molecules of retinal.
The two parts was joined together by strandard assembly, before it beta-carotene was amplified by Phusion PCR.
AraC-pBAD promoter comes from E.coli genome, while ctrEIBY are from Pantoea ananatis (formerly Erwinia uredovora) genome. Blh gene and its promoter are synthetized and codon optimezed by GeneScript.
Lactose/IPTG inducible promoter with RFP reporter tagged with DAS-ssrA degradation tag followed by terminator. The tagged RFP is degraded within cell, thus provides better temporal resolution of red fluorescence and promoter activity. Presence of SspB is crucial for effective protein degradation.
Part was assembled using BrickClip assembly (BBF RFC104) using other biobrick parts as templates. BrickClip assembly is a special case of more general Paperclip assembly method.[1]
Apis mellifera (honeybee) silk fibroin 3
This is the coding region for the honeybee silk protein #3 (will update more later)
Will update more later (mention that signal peptide has been removed)
Chaperone,the SicA???InvF complex activates transcription from the psicA promoter
This part is a highly repetitive DNA sequence which can bind TALE protein specifically.
This part is designed to contain two basic short sequences which can bind TALE1 and TALE2(3) specifically. These two basic sequences are repeated with no space by ten times, so it can bind more TALE protein and the spatial location of each pair of TALE1 and TALE2(3) is almost opposite.
This part is synthetized by viewsolid biotech firm (Beijing, China).
mRFP under control of copper responsive promoter
A copper iduceble promoter with RFP for detection
CopAp is a copper induceble promoter out of ``E.coli`` K12
Choi hiu Ying, LEE Cheuk Lam, Tse Yui Shing, Yeung Sin Yu
This sequence encoded for the delta15 desaturase. The gene is from Synechocystis sp. and optimised for the expression in E. coli. During the fatty acid conversion process, delta 15 desaturase catalyses the conversion of linolenic acid (18:2) into alpha-Linolenic acid, ALA (18:3) by adding a cis-doube bond at carbon 15.
The enzyme cut site must be designed according to iGEM prefix and suffix requirements.
The amino acid sequence of delta 15 desaturase is derived from Synechocystis sp. but codon optimised for the expression in E. coli.
Designed to close lacO present systems in higher temperatures by producing lacI.
The part is a composite part formed up of several distinct parts.
mammalian LacI with miR-373 binding site
In our project second step of cancer module is based on production of LacI so that it will suppress expression of DsRed. Binding site on 3' end of LacI makes it regulatable and limits it's expression only in cells with low miR-373 rate. In gastric epithelial cells level of this mirna is low but in gastric cancer cells level is very high.
We inserted two Mlu1 cut sites on each side of miR-373 binding so that other iGEM teams could easily replace it with any sequence.
This protein is mammalian codon optimized version of LacI repressor originally derived from E.coli. It also possess nuclear localization signal (NLS) so that it can act on lac operators.
Contactin-4-derived peptide binding to NADH Oxidase of S. pneumoniae
Short peptide of 13 amino acids corresponding to amino acids of the human protein Contactin-4 (isoform 3, UniProt Q8IWV2-3). It has been shown to bind to NADH oxidase (NOX) of Streptococcus pneumoniae and inhibit bacterial binding to human A549 cells (Muchnik et. al., 2013). Displayed on the surface of Bacillus subtilis, it should enable B. subtilis to bind specifically to S. pneumoniae. This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.
For generation of the part's nucleotide sequence, the amino acid sequence of the peptide (Muchnik et. al., 2013) was reverse transcribed with regard to the codon usage of Bacillus subtilis. Oligos were designed as precut by EcoRI and PstI to shorten the synthesized fragments and to simplify the cloning into pSB1C3.
The part was generated by annealing of artificially synthesized oligonucletides: C4P_ENX_SD_Ngo_precutEP_fwd: AATTCgcggccgctTCTAGAgtaaggaggaaGCCGGCATG TGGGAATGTAAAGCGAACGGAAGACCGAAACCGACATAT ACCGGTtaatACTAGTagcggccgCTGCA C4P_SNP_Age_precutEP_rev: GcggccgctACTAGTattaACCGGT ATATGTCGGTTTCGGTCTTCCGTTCGCTTTACATTCCCACATGCCGGCttcctccttacTCTAGAagcggccgcG
This is a engineered TAL effector that can bind with the DNA sequence 5'-GATAAACACCTTTC-3'
The part sequence should be able to correctly and efficiently bind with corresponding DNA, so we used bioinformatics tools to help us designing this sequence.
This part is built and standardized by ourselves using GATEWAY technique.
MaSp1 and MaSp2 12-mer block co-polymer in pSB1C3, with no regulatory elements. Ratio of 1 MaSp1 to 2 MaSp2.
Inducible generator of eYFP with C-terminal SH3 ligand
Lactose/IPTG inducible promoter wuth RFP reporter tagged with AAV-ssrA degradation tag followed by terminator. The tagged RFP is actively degraded within cell, thus provides better temporal resolution of red fluorescence and promoter activity. Part was assembled using BrickClip assembly (BBF RFC 104) using other biobrick parts as templates. BrickClip assembly is a special case of more general Paperclip assembly method.[1] Reference [1] Trubitsyna, M., Michlewski, G., Cai, Y., Elfick, A. & French, C. E. PaperClip: rapid multi-part DNA assembly from existing libraries. Nucleic Acids Res. 1???8 (2014). doi:10.1093/nar/gku829
The wss operon was extracted from P.fluorescens, an organism that produces cellulose acetate.
Modified rhlA coding sequence from Pseudomonas aeruginosa SQ6
RhlA together with RhlB encodes Rhamnosyltransferase I for mono-rahmnolipids biosynthesis in Pseudomonas aeruginosa.
A mutation has been done to remove the PstI restriction site contained in the coding sequence.
GFP (mut3b) with AAV-ssrA degradation tag
GFP (mut3b) (see part BBa_E0040) with added AAV-ssrA degradation tag (part BBa_I11012). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYAAAV and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The AAV tag is reported to lead to reasonably fast protein degradation, degrading GFP with rate -0.012 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations, protein stability, Km of binding to the protease, temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to RFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additonal residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (AAV in this case) are recognised by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
GFP comes from part BBa_E0040, tag sequence was obtained from part I11012, but same tag was also used in paper by Andersen et al., (1998).[2]
pRha - fimH 49 KO - RPMrel - SpyTag_225 - HisTag_225
Major Spidroin Protein 1 (MaSp1) with Sticky Ends AB
This regulatory part is made up of the Arabinose-Inducible promoter, pBAD, and its transcriptional inhibitor/activator, AraC. AraC inhibits gene expression in the absence of Arabinose by binding to the operator units present within the AraC and pBAD promoter sequences, and producing a DNA loop that prevents the RNA polymerase and CAP from binding and initiating transcription. When Arabinose is supplied, this binds to the AraC dimer and releases it from the operator site found in the pBAD promoter, at the same time it enhances transcription by allowing binding to the enhancer sites upstream.
This part was cloned out of existing templates (such as BBa_K325218, BBa_325219, all of which contain sfGFP downstream of the pBAD promoter)
pBar1: Mating factor alpha responsive promoter
The pBar1 promoter is a mating factor alpha responsive promoter found in MATa S. Cerevisiae. This promoter drives expression of Bar1, a protease that cleaves mating factor alpha.
We changed nucleotide 210 from thymine to cytosine. This does not change the amino acid Asparagine and is therefore a silent mutation. We also changed nucleotide 285 from cytosine to adenine. This does not change the amino acid valine and is therefore a silent mutation.
pBar1 is found in the genomic sequence of MATa S. cerevisiae.
fim switch[default OFF](wild-type)_rbs_gfp
This part is the coding sequence of the enzyme Indoleacetimide hydrolase Iaa H. This enzyme catalyses the hydrolysis of indoleacetamide to indoleacetate and ammonia. This part is gathered from the 2-step pathway in Pseudomonas savastanoi producing the auxin, indole-3-acetic acid.
In order to get the whole sequence of IAAH, we removed the RBS and other irrelevant sequences in K515100 when designing the PCR primers.
This part is gained from the part BBa_K515100 by PCR.
Artilysin Art-175 fused at N-terminal with DsbA signal peptide
Artilysins are an exciting class of enzyme-based antibacterials. Their name is derived from "artificial endolysin" and they exploit the lytic power of bacteriophage-encoded endolysins. Endolysins are peptidoglycan hydrolases produced at the end of the lytic cycle that pass through the cytoplasmic membrane, degrade the peptidoglycan layer and cause the osmotic lysis of the infected bacterial cell, thus liberating the progeny. Purified endolysins have been used to kill Gram-positive pathogens. Gram-negative bacteria, however, have a protective outer membrane containing lipopolysaccharide (LPS) that serves as a barrier against the peptidoglycan hydrolytic activity of endolysins from the outside. To overcome this problem, selected polycationic or amphipathic peptides that locally destabilize the LPS layer have been covalently fused to endolysins. Biers et al. fused the sheep myeloid antimicrobial peptide (SMAP-29) to the N terminus of the endolysin KZ144 to create Artilysin Art-175. Art-175 has been shown to be a highly potent antibacterial that acts in minutes to kill virtually all Pseudomonas aeruginosa strains. [2] BBa_K1659002 is a composite of Art-175 with the N-terminal DsbA signal peptide with a hexahistidine tag. DsbA targets recombinant proteins to the co-translational signal- recognition-particle (SRP)-dependent pathway to mediate secretion out of the bacterial chassis. [2]
Endolysin KZ144 originates from Pseudomonas aeruginosa bacteriophage varphiKZ. SMAP-29 originates from sheep genomic DNA.
The nadE gene encodes Naphthyl Acetamide synthetase which can boot up the NAD+(H) level in the cells facilitating the electron transferring rate in MFCs (Microbial Fuel Cells).
The sequence is a full length ORF containing the initiation codon and the termination codon.
This part is aquried from genome the E.coli K-12 substrain MG1655.
RBS + Antifungal GAFP-1 + Double terminator
Phytochelatin (PC) EC20 fused to linker-dCBD with T7 promoter
HisTag-Scaffold_Si4-Tag fusion protein with RBS BBa_B0034
RFP from Discosoma striata (coral) with AAV-ssrA degradation tag
Mutant RFP from Discosoma striata (coral) (see part E1010) with added AAV-ssrA degradation tag (part I11012). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYAAAV and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The AAV tag is reported to lead to reasonably fast protein degradation, degrading GFP with rate -0.012 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: 1) ClpXP and ClpAP protease and SspB mediator concentrations; 2) Protein stability; 3) Km of binding to the protease; 4) Temperature [3]. [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3]Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
RFP comes from part E1010, tag sequence was obtained from part I11012, but same tag was also usead in paper by Andersen et al., (1998).[2]
Lo Hiu Fung, Lau Tsz Kit, Chu Lok Ting, Wu Chun Ting
We have cloned a modified thioesterase gene, ???tesA, which codes for an E. coli thioesterase protein that lacks the N-terminal 26-amino acid signal peptide sequence, and is named here as ???leaderless TesA??? or ???TesA. The ???TesA protein is expected to be expressed exclusively in the cytosol of E. coli to catalyze the conversion of fatty acyl-ACP (acyl carrier protein) or fatty acyl-CoA to free fatty acids (Figure 1). ???TesA is predicted to enhance free fatty acid production in the genetically-modified E. coli strain.
The genome sequence of Escherichia coli K-12 substr. W3110 is available in GenBank at http://www.ncbi.nlm.nih.gov/gene/12930860.
MaSp2 3-mer in a T7-RBS expression vector (BBa_K552998)
sfGFP controlled by T7 promoter and arsenic operator
This part consists of sfGFP with a translation enhancing 5'-UTR under the control of the T7 promoter and arsenic operator. In combination with the E. coli arsenic repressor arsR, this part can be used as an arsenic biosensor.
This part was created by inserting the T7 promoter BBa_I719005 upstream of BBa_J33201 via 3A assembly. Subsequently, Gibson assembly was performed to replace arsR by sfGFP and introduce the UTR. The followoing primers and templates were used: Fragment 1: UTR_arsO_rev: ccttcttttttttttttaaagttaaacaaaattatTAAGTCAAAAACATATATGACTTAACG Cm_fwd: CGGCATCAGCACCTTGTC Template: pSB1C3-pT7-arsR Fragment 2: UTR_fwd: GTTTAACTTTAAAAAAAAAAAAGAAGGAGATATACATATGCGTAAAGGCGAAGAGCTGTTCAC Cm_rev: TATACGCAAGGCGACAAG Template: BBa_I746909
Based on three parts from the distribution.
Double CBD (dCBD) with N-terminal linker
RFP from Discosoma striata (coral) with LAA-ssrA degradation tag (wt)
Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LAA-ssrA degradation tag (part BBa_M0050). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is E. coli wild type ssrA tag and is reported to lead to very fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; Protein stability; Km of binding to the protease; Temperature [3]. Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LAA-ssrA degradation tag (part BBa_M0050). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is E. coli wild type ssrA tag and is reported to lead to very fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; Protein stability; Km of binding to the protease; Temperature [3]. Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LAA-ssrA degradation tag (part BBa_M0050). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is E. coli wild type ssrA tag and is reported to lead to very fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; Protein stability; Km of binding to the protease; Temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012)
RFP comes from part BBa_E1010, tag sequence was obtained from part BBa_M0050, but same tag was also used in paper by Andersen et al., (1998).[2]
Pseudomonas AutoInducer-1 response unit (luxR promoter)
Luna Barroco de Lacerda, Maria Clara Astolfi
The Bioaccumulative biobrick is composed of our ???essential biobrick??? (BBa_K1355001) + a Metallothionein (BBa_K346004) available in the database of the iGEM since 2010.In the presence of mercury, The MerR protein will bind to it and MerT, MerP and Metal Binding Protein (MBP) proteins are synthesized and bioaccumulation will start! The MerP and MerT protein are responsible for the transport of mercury from the periplasm to the cytoplasm, leading him to MBP! The MBP binds to the mercury in the cytoplasm, rendering it inactive.
For this construct, we followed these steps: Extraction of plasmid DNA and DNA quantification of bacteria transformed with the Essential Biobrick (BBa_K1355001) and the bacteria transformed with the MBP Biobrick (BBa_K346004); Verifying the electrophoretic profile of the extracted plasmid DNA; Restriction enzyme digestion for Essential Biobrick with SpeI and PstI and for MBP Biobrick with PstI and XbaI; Checking the electrophoretic profile of digested samples; Purification of the Fragment of interest from the gel; Ligation the gel purification of MBP gene and digested Essential Biobrick; Transformation of ligation in DH5-alpha; Plasmid DNA extraction from bacteria transformed; Check the electrophoretic profile to see results of samples linked (no fragments); Digestion of Essential Biobrick + MBP Biobrick with EcoRI and PstI, aiming to analyze the fragment size to be isolated; Checking the electrophoretic profile of the digested sample to obtain results showing that the isolated fragment is the junction of Essential Biobrick +MBP Biobrick ;
mammalian LacI with miR-223 and miR-21 binding sites
In our project second step of cancer module is based on production of LacI so that it will suppress expression of DsRed. Binding site on 3' end of LacI makes it regulatable and limits it's expression only in cells with low miR-223 and miR-21 levels. miR-21 has high rate of production in most cancer types. In gastric epithelial cells level of this miRNAs are low but in gastric cancer cells their level is very high.
We suggest also to view similar part we submitted-BBa_K1639010. It contains mLacI with miR binding sites but have restriction enzyme cut sites to remove miR binding site as needed.
This protein is mammalian codon optimized version of LacI repressor originally derived from E.coli. It also possess nuclear localization signal (NLS) so that it can act on lac operators in nucleus.
Major Spidroin Protein 2 (MaSp2) with sticky ends CA
This part contains the core sequence of MaSp2 that has been assembled with a 5'-CGTG-3' overhang on the 5' end of the sequence and 3'-TCAA-5' overhang on the 3' end of the sequence. This part was designed for use with ICA, along with parts BBa_K1763003 and BBa_K1763004 as an efficient method to assemble multiple MaSp2 sequences together. This setup is to show that this particular set of sticky ends is suitable for ICA. Before using in ICA, this part should be digested with BsaI, a type IIs restriction enzyme, which cuts DNA outside of its recognition site.
gabR gene and gabT promoter from Bacillus subtilis
This is a part of the gab operon of Bacillus subtilis. It includes the gene for the repressor protein GabR under the control of its natural promoter as well as the gabT promoter, which is regulated by GabR. GabR is inactivated by γ-aminobutyrate(GABA). The expression of any gene that is inserted downstream of this part can therefore be induced by GABA.
The coding sequence of gabR is codon optimized for expression in E. coli.
This part was obtained by gene synthesis.
Prefix homologous recombination for Synechocystis ("Left flank")
This is a prefix region of DNA for homologous recombination into a neutral site within the genome of Synechocystis sp. PCC 6803. Kunert, A., Hagemann, M. & Erdmann, N. (2000) Construction of promoter probe vectors of Synechocystis sp. PCC 6803 using the light-emitting reporter systems Gfp and LuxAB. J. Microbiol. Methods. 41, 185-194 Angermayr, S. A., Paszota, M. & Hellingwerf, K. J. (2012) Engineering a cyanobacterial cell factory for production of lactic acid. Appl. Environ. Microbiol. 78, 7098-7106
This part comes from genomic sequence of Synechocystis sp. PCC 6803
Synechocystis prefix homologous integration region
Dye-decolourising peroxidase from Bacillus subtilis
BsDyP gene (1251bp) from Bacillus subtilis that has been found to be effective in degrading lignin and azo dyes, in an oxidative process.
From genomic sequence of Bacillus subtilis; BsDyP gene inserted into a pET-21a (+) expression vector, provided to us by XXX Research Group.
including FKBP, 2??linker, and firefly luciferase C terminal 394 amino acid domain.
FKBP and C terminal 394 amino acid domain are linked by 2??linker
C394 terminal domain of firefly luciferase
NADPH-dependent flavinoid reductase relate to Met-DMSP byosynthesis pathway(REDOX)
This part is NADPH-dependent flavinoid reductase gene of Fragilariopsis cylindrus. The gene code NADPH-dependent flavinoid reductase relate to Met-DMSP byosynthesis pathway from Fragilariopsis cylindrus. which catalyze second step of Met-DMSP biosynthesis pathway.
tsPurple Reporter + 3-alpha-HSD (RBS + Coding + Terminator)
pSB1C3-6H-dCas9-F3, this is the fusion protein of dCas9-F3, His tag allows for protein purification
The coding sequence of this fusion protein is too long, you'd better amplify is separately.
Microcin S, a Class II microcin produced by probiotic E. coli G3/10
Microcins are antibacterial peptides. Microcin S was isolated from Escherichia coli G3/10, a component of the probiotic drug Symbioflor 2. Here it was shown to be responsible for E. coli G3/10's ability to suppress adherence of enteropathogenic E. coli E2348/69. [1] The Microcin S operon is about 4.7 kb in size and is comprised of four genes. This part is the sequencing encoding the microcin itself (mcsS). [1] Zsch??ttig, A., Zimmermann, K., Blom, J., Goesmann, A., P??hlmann, C., & Gunzer, F. (2012). Identification and characterization of microcin S, a new antibacterial peptide produced by probiotic Escherichia coli G3/10. PLoS ONE, 7(3), 1???9. http://doi.org/10.1371/journal.pone.0033351
superfolder GFP with N-terminal pdu-microcompartment tag
We have used superfolded GFP with the N-terminal pdu-microcompartment tag in order to get fluorescence images that would show localised spots of higher GFP-density, indicating that the microcompartment tag successfully inserts the attached protein into the microcompartments
We have designed Primers to amplify sfGFP from a plasmid, provided by Dr. Ciaran Kelly, in order to attach the microcompartment tag upstream of the sfGFP-coding region.
We received a sample of sfGFP in a holding vector, provided by Dr. Ciaran Kelly.
pSB1C3-6H-dCas9-Clact, C terminal segment of beta-lacatamse fused with dCas9, His tag allows for protein purification
delta alpha segment of LacZ fused with dCas9, His tag allows for protein purification
This part required for producing reporter flourescence protein GFP from Toehold-GFP(BBa_K1639000) composite part.
This part synthetase with strong T7 Promoter downstream of Trigger RNA. There is also a T7 Terminator after Trigger RNA.
Wyss Institute for Biologically Inspired Engineering. Sequence is taken from supplementary data of article: "Toehold Switches: De-Novo-Designed Regulators of Gene Expression". (PMID: 25417166)
J23100 + β-carotene 15,15’-dioxygenase (blh gene)
blh encodes for β-carotene 15,15'-dioxygenase, the enzyme that cleaves one molecule of β-carotene into two molecules of retinal
The part was subcloned from pUC57 into pSB1C3
The sequence of uncultered cluster bacteria SAR86 was synthetized and codon optimized by GeneScript
Synthetic Phytochelatin EC(20) rfc25 fusion format
Sequence is not natural. Basis for the sequence was from the following reference: Bae W, Chen W, Mulchandani A, Mehra RK (2000) Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnol Bioeng. 70(5):518-24.
Cis-aconitate decarboxylase (cadA) from Aspergillus terreus
RBS + NpuDnaE N-intein RFC[105] non-splicing control construct (with His6)
Sortase A non-circularization construct (with Smt3 and His6; without RBS)
pSB1C3-6H-F3-dCas9, fusion protein of F3 segment of DHFR fused with dCas9, the His tag facilitates protein purification
Major Spidroin Protein 1 (MaSp1) with Sticky Ends BC
This part contains the core sequence of MaSp1 that has been assembled with a 5'-TGTC-3' overhang on the 5' end of the sequence and 3'-GCAC-5' overhang on the 3' end of the sequence. This part was designed for use with ICA, along with parts BBa_K1763003 and BBa_K1763004 as an efficient method to assemble multiple MaSp sequences together. This setup is to show that this particular set of sticky ends is suitable for ICA. Before using in ICA, this part should be digested with BsaI, a type IIs restriction enzyme, which cuts DNA outside of its recognition site.
Mercury responsive promoter with UTR-sfGFP
mercury responsive promoter with 5??untranslated region and sfGFP for detection
RBS + SspDnaB N-intein RFC[105] assembly construct (with His6)
MaSp1 and MaSp2 12-mer block co-polymer under control of T7 promoter and RBS. Ratio of 2 MaSp1 to 1 MaSp2.
fim switch[deault ON](Tokyo_Tech/J23119)
Dispersin B fused at N-terminal with DsbA 2-19 signal sequence
Supernova is the monomeric version of KillerRed (BB_K1184000). Codon optimized Supernova with WTLac promoter allows for induction of Supernova expression with IPTG.
The promoter Plux in induced by the dimer AHL-luxR. As a result, amilCP, a blue chromophoire protein, is synthesized. TetR is a repressor of the promoter Ptet.
BBa_R0062: Plux, BBa_B0030: Strong RBS, BBa_K592009: amilCP, BBa_B0010:T1 (terminator) from E. coli.
fim switch[default OFF](Tokyo_Tech/J23119)_rbs_gfp
This is a engineered TAL effector that can bind with the DNA sequence 5'-CTTTCCACAAATAG-3'
T7+ ibPB RNA Thermometer + pelB + MCS + 6xHis
The part is to act as a backbone for the T7+ibPB RNA Thermometer+pelB+6xHis system. The MCS(Multiple Cloning Site) as the ligation region for the parts to be integrated into the system. The part that is added to the backbone will be translated in the presence of higher temperatures. The pelB signal peptide is present for the produced protein to be carried out of the cell. The 6xHis is for protein analysis.
This is a composite part formed up of distinct parts from distinct origins.
His-tagged arsenic repressor fused to superfolder GFP under the control of T7 promoter
Uses T7 promotor and a strong rbs (BBa_K525998), the arsenic repressor linked with superfolding GFP (sfGFP) with a His-Tag. By linking sfGFP we want to detect bound protein via fluorescence.
We clone arsR in T7-RBS-sfGFP and added a His-Tag via PCR and Gibson Assembly.
This is a composite part which enables a high protein expression and an easy purification of arsR fused to superfolding GFP (sfGFP). The green fluorescence of the culture is a sign for protein expression.
CBD2 is EnLinker-CBH2-EnLinker-CBH1 from T.reesei. TBC
codon optimized PAL with T7 promoter and Flag Tag
This part is the codon optimized version of KillerRed (BBa_K1184000). It has been optimized for E. coli by eliminating the rare codons.
Increase the functionality of KillerRed through codon optimization.
This part was codon optimized from the original KillerRed (BBa_K1184000)
fim switch[default ON](Tokyo_Tech/R0010)
This is a functional coding sequence for the protein NixA, which is a nickel transporter from H. pylori. This part will result in nickel intolerance from higher levels of intracellular nickel.
The Anderson promoter provides strong constitutive expression of NixA in E. coli.
Anderson promoter is that of part BBa_J23102. Terminator is that of part BBa_B1006. NixA gene is naturally found in H. pylori. This part was synthesized by GenScript in the vector pUC57. Part was then transferred into pSB1C3 using standard cloning procedures.
In our project we needed to express two different genes at the same bacteria, but unfortunately because of high repeats of sequences gene synthesizing company couldn't synthesize it. To solve this we just divided our genes into different plasmids and cotransformed it into same bacteria. For coexpression of genes from different plasmids origins have to be compatible, like ColA and ColE1. pSB1C3 possess ColE1 origin of replication. So it's compatible for expression with plasmid containg ColA origin of replication. But it doesn't contain expression machinery like promoters, terminators. We inserted this part to modify pSB1C3 so that it can be used as expression vector.
We inserted BamH1 and Sal1 cut sites between T7 promoter and T7 terminator. This part also contain RBS. Any other team can use this part to modify vectors like pSB1C3 to add T7 expression feature. Just clone it with enzymes at prefix and suffix.
It is combination of T7 promoter-terminator with sequences containing restriction enzyme cut sites.
FP:GTTTCTTCGAATTCGCGGCCGCTTCTAGATTGACTCTGTAGTTGCTACA VP:GTTTCTTCCTGCAGCGGCCGCTACTAGT ATTGCACACC CTGTAGCAAC TACAGA
This part is a fusion of the Apis mellifera (Western Honey Bee) silk protein coding region and the superfolder GFP coding region. A T7 promoter regulatory element has been added upstream, along with a ribosomal binding site. (in progress)
Subunit of a 12-subunit protein cage with a thrombin cleavage sequence inserted between residues 243 and 248 (ALPSAE to ALPGLVPRGSGSAE).
Considered the area to insert the cleavage sequence. This mutation was selected by examining the secondary structure to ensure minimal disruption of existing alpha-helices or beta sheets, sites were sandwiched by glycine residues, and used PyMOL to check if the site would be accessible to the protease. Geometric accessibility by the protease thrombin and minimal speculated extent of conformational changes by insertion.
Bromoperoxidase (trimer) and M1 Virus Matrix Protein (dimer) linked with an alpha helix.
Chromium responsive promoter under T7-promoter with UTR-sfGFP
T7 controlled Chromium responsive promoter with 5??untranslated region and sfGFP for detection
gRNA for SaCas9 targeting human ubiquitin B gene
Sascha Hein, Daniel Sachs, Christian Sator
We construct a lactate sensing system for our MFC.
Keep special enzyme site to change component of the system in order to regulate parts' expression level.
Artilysin Art-175, an antibacterial fusion protein
including T7 promotor, LacZ a', crRNA (tracrRNA) and T7 terminator.
Split sfGFP reconstitution N-terminal non-splicing control half
This part is another copy of the RBS30 (BBa_B0030). It is a Strong RBS based on Ron Weiss thesis.
The reason why we built this part by ourselves is that the copy of B0030 in the 2015 DNA distribution we received is sequenced as incorrect. And same problem also occurred in several other teams.
The part sequence was gained from the registry, the part is annealed from the ssDNA synthesized by Life(R).
Sortase A non-circularization construct (with His6)
RBS + SspDnaX C-intein RFC[105] assembly construct (with His6)
mRFP with 6x His tag (BBa_K1491008) and a strong RBS (BBa_B0034). It can be used as a reporter.
The tRNA synthetase/tRNA needed for incorporating o-(2-nitrobenzyl)-L-tyrosine (ONBY) at a UAG codon
The part includes the gene coding for the Methanocaldococcus jannaschii synthetase mutated to charge the orthogonal tRNA (also in the part) with the non-canonical amino acid o-nitrobenzyl tyrosine (ONBY) along with the proper promoters and terminator . This part encodes for the incorporation of ONBY during translation at AUG amber stop codons in a cell with Release Factor 1 (RF-1) knocked out such as "Amberless" E. coli or "RF-0".
Evolved from the Methanocaldococcus janaschii tyrosyl synthetase and tRNA by the Schulz group. Specific mutations sourced from Deiters, A., Groff, D., Ryu, Y., Xie, J. and Schultz, P. G. (2006), A Genetically Encoded Photocaged Tyrosine. Angew. Chem. Int. Ed., 45: 2728???2731.
Converts the amino acid tryptophan into 5-HTP, which is a precursor to serotonin. Soon to be edit
Optimised for E.coli, removed illegal restriction enzyme sites
including firefly luciferase C terminal 398 amino acid domain, 2??linker, and FKBP.
FKBP and C terminal 398 amino acid domain are linked by 2??linker
Expa4 plant secretion signal, localizes to extracellular space
Coding region for first 20 aminoacids of plant protein Exansin4. Secretion signal for plants (derived from A. thaliana). When fused to a protein, directs the protein to extracellular space.
Derived from Expansin4 from Arabidopsis thaliana(NP_181500).
Modified rhlB coding sequence from Pseudomonas aeruginosa SQ6
RhlB together with RhlA encodes Rhamnosyltransferase I for mono-rahmnolipids biosynthesis in Pseudomonas aeruginosa. This part includes bp upstream noncoding gene and bp downstreanm gene of RhlB. Two mutations have been done to remove the PstI restriction sites contained in the coding sequence, which won't change the amino acid sequence.
Two mutations have been done to remove the PstI restriction sites contained in the coding sequence, which won't change the amino acid sequence.
RFP from Discosoma striata (coral) with DAS-ssrA degradation tag
Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added DAS-ssrA degradation tag (part BBa_M0052). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYADAS and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The DAS tag is reported to have low affinity to ClpX thus its mediated degradation very much depends on the concentration of SspB (ClpX mediator).[1] However, be aware that exact protein degradation rate is influenced by multiple other factors: ClpXP and ClpAP protease concentrations, protein stability, Km of binding to the protease, temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
RFP comes from part BBa_E1010, tag sequence was obtained from part BBa_M0052, but same tag was also used in paper by Andersen et al., (1998).[2]
RBS + Gp41-1 N-intein RFC[105] assembly construct (with His6)
gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm under U6 promoter
The promoter Plux in induced by the dimer AHL-luxR. As a result, amilCP, a blue chromophoire protein, is synthesized. Lambda phage CI is a repressor of the promoter PCI.
The OprF is a major outer membrane protein of Pseudomonas aeruginosa. This protein functions as a nonspecific porin to allow the passage of small hydrophilic molecules, plays a structural role in maintaining cell shape and outer membrane integrity, and is required for growth under low osmolality. The structure of OprF has been proposed to consist of three domains, the N-terminal forming h-barrel structure, a loop or hinge region, and the C-terminal associated with peptidoglycan. Based on the predicted secondary structure and information found in the literature, we chose Ala196 as potential fusion sites for displaying CBP. CBP is the abbreviation of copper binding peptide made up of seven amino acid.
Based on the predicted secondary structure and information found in the literature, we chose Val(188) as potential fusion sites for displaying CBP. CBP is the abbreviation of copper binding peptide made up of seven amino acid.
The OprF gene is from Pseudomonas aeruginosa.
Sortase A non-circularization construct (with His6; without RBS)
A right promoter which can sense formaldehyde while there is a left promoter which can erpress the HxlR protein theoretically.The HxlR protein can enhance the function of the P-formaldehyde.
The start codon of the HxlR protein is UUG.
We get this part from Bacillus subtilis 168.
beta-Neurexin-derived peptide binding to S. aureus adhesin SdrC
Short peptide of 11 amino acids corresponding to the N-terminus of the human protein neurexin beta-1. It includes the binding site (HHIHHFH) for the Staphylococcus aureus adhesin SdrC. Displayed on the surface of Bacillus subtilis, it shoud enable B. subtilis to bind specifically to S. aureus.
Oligos were designed as precut by EcoRI and PstI to shorten the synthesized fragments and to simplify the cloning into pSB1C3.
The part was generated by annealing of artificially synthesized oligonucletides: Nrx1b_ENX_SD_Ngo_precutEP_fwd: AATTCgcggccgctTCTAGAgtaaggaggaaGCCGGCATG GCGCACCACATTCACCACTTTCACGGATCA ACCGGTtaatACTAGTagcggccgCTGCA Nrx1b_SNP_Age_precutEP_rev: GcggccgctACTAGTattaACCGGT TGATCCGTGAAAGTGGTGAATGTGGTGCGC CATGCCGGCttcctccttacTCTAGAagcggccgcG
This promoter can sense the whether there is formaldehyde or not.It can be activitated by formaldehyde with the help of the protein hxlR.This promoter may have some leakage in the strain E.coli DH5a .
There may be different expression levels when you use it in different stains of E.coli.
This part comes from the Bacillus subtilis genomic sequence.
Protein scaffold with N-terminal HisTag and RBS BBa_B0034
MerR activator under constitutive promoter and induceble merT promoter with 5 UTR -sfGFP
eYFP with C-terminal GBD ligand and RBS BBa_B0034
This is a engineered TAL effector that can bind with the DNA sequence 5'-GGAGGCACCGGTGG-3'
This part is built by NJU_CHINA using GATEWAY technique. We standardized this part by ourselves.
Coding for the trans-acting regulator SmtB
In 1993, Huckle and co-workers reported the isolation and analysis of the smt locus from Synechococcus sp.. Gene smtB, one of the components of the locus, encodes a transcriptional repressor of gene smtA, with which is required for normal tolerance to Zn2+ and Cd2+. The product of smtB functions by binding to the smt operator-promoter(smtO-P) and dissociating in the presence of Zn2+ due to the classical helix-turn-helix motif similar to many DNA-binding proteins. It can also be genomically rearranged when induced by Cd2+, leading to a deletion within smtB because of a highly iterated palindrome (HIP1), a highly represented octanucleotides(5′-GCGATC-GC-3′) which traverse both borders of the excised element. These two mechanisms both cause the elevated expression of smtA.
According to the mechanisms of smt locus represented above, gene smtB should function with the accompany of smt operator-promoter. Previous cases selected smtA whose product functions by encoding MT-like to sequester metal ions, while our case intended to take advantage of the trigger lies in this locus. By imitating former researches, in which a promoter less lacZ gene was fused upstream of smtA, we respectively splice two selected reporter genes and sequence from smt operator-promoter to smtB, equivalent to replacing smtA with reporter genes, to visualize the effects of metal ions on the locus.
Metallothioneins(MTs), discovered in horse kidney in 1957 by Margoshes and Vallee are identified as low-molecular weight and sulphydryl rich proteins which bind metal ions in metal-thiolate clusters and whose synthesis increases in response to elevated concentrations of certain metals. In 1988, Olafson and co-workers reported the amino acid sequence of a metal-associated protein isolated from cyanobacterium Synechococcus sp.. With the function of binding metal ions remaining, it only has less than 20% homology in amino acid sequence with known MTs in eukaryotes and thus we call it class II MT or on some occasions MT-like. Owing to the works of Robinson and Huckle, the corresponding locus of Synechococcus PCC7942, smt locus was isolated and analyzed. It includes smtA which encodes the class II MT and a divergently transcribed gene smtB which encodes a trans-acting, autoregulatory repressor that exerts metal ion-inducible negative control over smtA transcription. Between them lies a 100-bp operator-promoter region(smt O-P, BBa_K1509001), which is responsible for the divergent orientation of smtA and smtB.
This is the coding region of a small antifreeze protein. It works by binding to the ice crystals as they form inside the cell, and by doing so prevents the ice crystals from enlarging and penetrating the cell membrane. We wanted to test if there would be a significant difference in the growth rate of moss, Physcomitrella patens, with the antifreeze gene, compared to the one without. This would be done at a low temperature to test the in-vivo function of the antifreeze protein.
We used codon optimization for moss so it would work better in Physcomitrella Patens.
The part is taken from a spruce budworm, Choristoneura fumiferana, living in areas with permafrost.
Deinococcus radiodurans UV DNA damage endonuclease - uvsE
TODO - mention the different starting points
Deinococcus radiodurans R1 genomic DNA from Chromosome 1
GoldenGate compatible Green Flourescent Protein (GFP)
A variation of the part BBa_E0040 that has been made compatible for Golden Gate by removal of internal BsaI and BpiI recognition sequences. Fluoresces under UV light when the gene is expressed.
Domestication for GoldenGate by removal of internal BsaI and BpiI recognition sequences.
This part is sensitive to long chain acyl-CoA ,with a FadR binding site. In the absence of long chain acyl-CoA, FadR represses the transcription. In the presence of long chain acyl-CoA, FadR dissociates from the binding site so that the transcription begins. E.coli uses this promoter to regulate fatty acid metabolism.
Dispersin B, an antibiofilm enzyme that specifically hydrolyses PGA polymers
Myristoilation signal sequence as RFC[105] N-terminal insert
The promoter Plux is induced by the binding of an AHL-luxR dimer. This bio-brick leads to the synthesis of the enzyme Green Renilla Luciferase, which catalyses a luminescent reaction. TetR can be used to repress the promoter Ptet.
Supernova is the monomeric version of KillerRed (BBa_K1184000). It has been codon optimized for E. coli.
The original KillerRed is BBa_K11840000.
We incorporated tag1 into a RFP-based test platform for inducible protein degradation in E. coli.
signal sequence from USP45 fused to aiiA
- one nucleotide in the signal sequence of USP45 had to be changed because it contained the illegal restriction site PstI
-aiiA was isolated out of the biobrick BBa_C0060. -ssUSP45 was isolated from the synthetic gene ssUSP45DspB made by IDT.
RhlI Acyl-homoserine-lactone synthase from P.aeruginosa SQ6
This sequece encodes RhlI, an acyl-homoserine-lactone synthase required for the synthesis of BHL (N-butanoyl-L-homoserine lactone), and HHL (N-hexanoyl-L-homoserine lactone) autoinducer molecules which bind to RhlR.
It includes both the complete coding sequence of RhlI and part of noncoding sequence between RhlR and RhlI gene.
It comes from the genomic sequence of P.aeruginosa SQ6.
The M. florum ssrA tag (mf-ssrA) is degraded by its endogenous Lon protease (mf-Lon) but not by E. coli Lon or ClpXP, and mf-Lon does not recognize or degrade ec-ssrA, providing a protease and cognate degradation tag with orthogonal functionality in E. coli.
global DNA-binding transcriptional dual regulator H-NS
This part contains an additional BamHI restriction site at beginning of TlpB sequence and a XhoI restriction site at the end.
Escherichia Coli K-12, sequence taken from Genbank: http://www.ncbi.nlm.nih.gov/gene/945829
RBS + NpuDnaE C-intein RFC[105] non-slicing control construct (with His6)
UTR-sfGFP controlled by T7-promoter and lead responsive promoter
lead induceble promoter under the control of T7 Promoter with mRFP for detection
This part consists of T7 promoter lead induceble Promoter PbrA from ''Cupriavidus metallidurans CH34'' and mRFP for detection
This part consists of B0034 RBS, human choline acetyltransferase (CHAT) gene with C-terminal His tag, and B0015 terminator.
- human CHAT gene (UniProt accession no: P28329) was codon-optimized for E.coli and provided with C-terminal 6xHis tag - due to its length, the sequence was synthesized as two gBlock fragments with 30 bp overlap between each other and with 20 bp overlaps with respective PSB1C3 ends - the construct was cloned into PSB1C3 using Gibson Assembly
Lead repressor under control of constitutive promoter and strong RBS and lead responsive promoter wi
Repressor for lead responsive promoter under the control of konstitutive promoter and lead responsive promoter with 5??untranslated region and sfGFP for detection. As a lead biosensor
genomic sequence out of ????Cupriavidus metallidurans???? CH34
Split sfGFP reconstitution C-terminal non-splicing control half
MaSp1 and MaSp 12-mer block co-polymer under control of T7 promoter and RBS
Major Spidroin Protein 2 (MaSp2) Sequencing Core with AB Sticky Ends
This part contains the core sequence of MaSp2 that has been assembled with a 5'-AGTT-3' overhang on the 5' end of the sequence and 3'-ACAG-5' overhang on the 3' end of the sequence. This part was designed for use with ICA, along with parts BBa_K1763003 and BBa_K1763004 as an efficient method to assemble multiple MaSp2 sequences together. This setup is to show that this particular set of sticky ends is suitable for ICA. Before using in ICA, this part should be digested with BsaI, a type IIs restriction enzyme, which cuts DNA outside of its recognition site. This part codes for identical protein primary sequence as BBa_K17630002, but has a different DNA sequence. This part is intended to anneal to a sequencing primer that will allow longer composite MaSp genes to be read.
mOrange with 6x His tag(BBa_K1491006) and a strong RBS (BBa_B0034). It can be used as a reporter.
RBS is BBa_B0034 and mOrange is BBa_E2050
cell division inhibitor of the MinC-MinD-MinE and DicB-MinC systems that regulate septum placement
Codon optimized KillerRed with WTLac promoter allows for induction of KillerRed expression with IPTG.
Hammerhead Ribozyme and Malchite Green Aptamer in BBF RFC 110 transcription cassette
Hammerhead Ribozyme (HHR) and Malchite Green (MG) Aptamer in BBF RFC 110 transcription cassette for live tracking of in vitro transcription.
Cloned without restriction enzymes to avoid issues occurring from conserved cut sites. For more details see BBF RFC 110.
Synthetic, Malchit Green aptamer (Grate, D., and Wilson, C. (1999). Laser-mediated, site-specific inactivation of RNA transcripts. Proceedings of the National Academy of Sciences of the United States of America 96, 6131-6136.) HHR (Prody, G.A., Bakos, J.T., Buzayan, J.M., Schneider, I.R., and Bruening, G. (1986). Autolytic processing of dimeric plant virus satellite RNA. Science 231, 1577-1580)
The clp gene from Xanthomonas campestris
c-di-GMP Repressed DNA Binding Protein (Clp) Quorum sensing and bacterial cell signalling are mechanisms known to be utilised by bacteria to regulate their virulence. The RpfC-RpfG sensory system has been found to activate the production of virulence factors like extracellular enzymes and polysaccharides in Xanthomonas campestris. It has been also suggested that this system is involved in linking environmental stimuli, including DSF, with pathogenicity in Xanthomonas campestris. DSF is a cis-unsaturated fatty acid from the DSF family. The RpfC-RpfG two-component system is the most well understood one and is present in all Xanthomonads. http://www.ncbi.nlm.nih.gov/pubmed/?term=r+ryan+dsf+family http://www.ncbi.nlm.nih.gov/pubmed/?term=A+two-component+system+involving+an+HD-GYP+domain+protein+links+cell%E2%80%93cell+signalling+to+pathogenicity+gene+expression+in+Xanthomonas+campestris
The sequence had 1 PstI site that was mutated out by site-directed mutagenesis.
CsgA under control of L-rhamnose-inducible promoter
The nucleotides encoding for the second and third amino acid of the CsgA gene were changed to optimize synthesis success. (silent mutations)
This part was synthesized. The rhamnose promoter was used by the iGEM 2014 TU Delft teama and originally added by the iGEM12 Paris Bettencourt team. RBS and CsgA originate from E. coli K-12 MG1655
SuperYFP2 with 6x His tag(BBa_K1491004) and a strong RBS (BBa_B0034). It can be used as a reporter.
RBS is BBa_B0034 and SuperYFP2 is BBa_K864100