Christian Sator,Stefan Zens, Max Zander, Benedikt Spannenkrebs, Sebastian Jaeger
Andrew D Halleran, Michael C LeFew, Joseph L Maniaci, John P Marken, Caroline A Golino
Cyan fluorescent protein with LVA fast degradation tag under the control of pBad promoter I13453.
Dispersin B, an antibiofilm enzyme that specifically hydrolyses PGA polymers
Leon Kraus, Jonathan Pletzer-Zelgert, Jonas Sindlinger, Carmen Sakas Gandullo, Sebastian Holzner, Elena Tonchevska, Mirjam Haun
Biosensor device for detection of GHB and GBL
This part serves as a generator of sfGFP when induced.
Ste2: ligand receptor of mating factor alpha
Ste2 is an integral ligand receptor of alpha factor found endogenously in MATa S. Cerevisiae (budding yeast).
Nucleotide 1104 changed from T to C to remove PstI cutsite; silent mutation
Ste2 is found in the genomic sequence of MATa S. Cerevisiae.
Anna Fomitcheva, Lisa Baumgartner, Charlotte Ramon
Collection of promoters regulated by LldR and LacI.
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]
Dispersin B fused at N-terminal with DsbA 2-19 signal sequence
LsrC of LuxS/AI-2 signaling pathway in Salmonalla
Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. And autoinducer-2 (AI-2) has been proposed to serve as a 'universal signal' for interspecies communication. In the LuxS/AI-2 signaling system of Salmonella Typhimurium, AI-2 response involves ATP binding cassette transporter encoded by genes named Lsr (LuxS regulated). And LsrC is thetransmembrane protein which constructed the ABC transport. In our project, we set this protein-coding part under a nisA promoter and try to integrate them in the genome of Lactobacillus or Lactococcus for the final purpose of constructing an integrated AI-2 response pathway of Salmonella in the engineered bacteria.
The lsrC gene was isolated from the genome of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 using PCR. We added restriction enzyme sites when designing primers, and the assembly is done through the use of restriction sites (cutting and ligating).
We extracted the genomic DNA of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, and used PCR to isolate the gene LsrA(Gene ID: 1255602).
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.
including T7 promotor, LacZ a', crRNA (tracrRNA) and T7 terminator.
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 responsive promoter with mRFP for detection
lead responsive promoter of ????Cupriavidus metallidurans???? CH34
T7 Promoter + Ribosome binding site + EcnB + 6x Hist tag + terminator. Coding device for anti-microbial peptide derived from Escherichia coli EcnB.
Use in conjunction with strain-expressing T7 polymerase
Synthesized by Integrated DNA Technologies
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 MaSp 12-mer block co-polymer under control of T7 promoter and RBS
Coding sequence for nitric oxide-sensitive repressor of pYeaR.
This part was synthesized from E. coli genomic data retrieved via EcoGene.
This is a coding sequence of C. parapsilosis mating pheromone receptor. Cells with this receptor being expressed react to the C. parapsilosis mating pheromone by the activation of the pheromone mating pathway. The coding sequence was codon optimized for the expression in S. cerevisiae. C-terminal was replaced by a C-terminal from C. albicans for more efficient coupling to the mating pathway in S. cerevisiae. Detailed description can be obtained from the [http://2015.igem.org/Team:Czech_Republic/Project/Synthetic_signals_and_receptors#Receptors iGEM 2015 Czech Republic wiki].
The coding sequence was codon optimized for the expression in S. cerevisiae. Kozak sequence was added before the start codon for a better translation. C-terminal was replaced by a C-terminal from C. albicans for more efficient coupling to the mating pathway in S. cerevisiae.
the coding sequence of the fusion protein of dCas and the CFluc of the luciferase
Jonas Sindlinger, Leon Kraus, Jonathan Pletzer-Zelgert, Elena Tonchevska, Sebastian Holzner, Mirjam Haun, Sebastian Barthel
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 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.
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 located upstream of the LsrC open reading frame (inside the LsrA open reading frame) in E. coli
This part was synthesized, and is based on E. coli genomic data retrieved via EcoGene: http://www.ecogene.org/gene/EG13809
LIP prepro + E. coli ribosomal protein L2 (1-60,GS linker,202-273aa) + YLcwp3
__NOTOC__ BBa_K1592008 short 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. Here we use this system to display Silica-tag on cell wall of Yarrowia lipolytica to binding the silica. 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, called Si-tag1, Si-tag2 and Si-tag3. We respectively test the silica-binding characteristics of this three regions and their combinations.
In order to replace the domains more conveniently, we respectivey added BamHI, SalI, NdeI between LIP2 prepro, E. coli ribosomal protein L2, GS linker and YLcwp3
E. coli ribosomal protein L2 was synthesized by IDT, LIP2 prepro and YLcwp3 were cloning from the plasmid JMP62, and we fused this three sequences.
Lead repressor under control of constitutive promoter and strong RBS
Lead promoter repressor under the control of konstitutive promoter (K608002)
Artilysin Art-175, an antibacterial fusion protein
LIP prepro + E. coli ribosomal protein L2 (1-60,203-273aa) + YLcwp3 Fusion
Collection of promoters regulated by LldR.
fim switch[default OFF](Tokyo_Tech/J23119)_rbs_gfp
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.
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]
MaSp1 and MaSp2 12-mer block co-polymer in pSB1C3, with no regulatory elements. Ratio of 1 MaSp1 to 2 MaSp2.
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
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
Stefan Robert Marsden, Max van 't Hof, Samantha Basalo Vazquez
CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms taken from E.coli K-12 MG1655. The aggregation to the nanowire ('curli') is induced by the membrane protein CsgB. CsgC, CsgE, CsgF and CsgG act as chaperones for CsgA during translation and export to the extracellular space. This part was designed to measure intracellular expression rates of CsgA coupled to fluorescence (GFP) by cloning the biobrick I13504 into the same operon which is under control of the Rhamnose promoter.
The scar is a stop codon, but no terminator. Like this, cloning a GFP which contains an RBS we intended to directly couple CsgA expression to expression of GFP.
This part was synthesized. The rhamnose promoter was used by the iGEM 2014 TU Delft team and originally added by the iGEM12 Paris Bettencourt team. RBS and CsgA originate from E. coli K-12 MG1655
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
LsrB of LuxS/AI-2 signaling pathway in Salmonalla
Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. And autoinducer-2 (AI-2) has been proposed to serve as a 'universal signal' for interspecies communication. In the LuxS/AI-2 signaling system of Salmonella Typhimurium, AI-2 response involves ATP binding cassette transporter encoded by genes named Lsr (LuxS regulated). And LsrB a solute-binding protein which could transport the AI-2 into the cytoplasm. In our project, we set this protein-coding part under a nisA promoter and try to integrate them in the genome of Lactobacillus or Lactococcus for the final purpose of constructing an integrated AI-2 response pathway of Salmonella in the engineered bacteria.
The lsrB gene was isolated from the genome of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 using PCR. We added restriction enzyme sites when designing primers, and the assembly is done through the use of restriction sites (cutting and ligating).
We extracted the genomic DNA of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, and used PCR to isolate the gene LsrA(Gene ID: 1255604).
Aldose reductase (GRE3) from Saccharomyces cerevisiae S288c
Alexandra Goretzki, Tanja Habeck, Nina Kuschik-Maczollek, Laurin Monnheimer, Steven Pilger, Bianca Reisinger, Daniel Stumpf, Sebastian Barthel
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.
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.
Sebastian Barthel, Niklas Hummel, Jasmin Weber, Michelle Zoeller, Marcus Geissler, Stefan Bohn, Simon Linder
LIP prepro + E. coli ribosomal protein L2 (1-273aa) + YLcwp3
Long description: __NOTOC__ BBa_K1592008 short 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. Here we use this system to display Silica-tag on cell wall of Yarrowia lipolytica to binding the silica. 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, called Si-tag1, Si-tag2 and Si-tag3. We respectively test the silica-binding characteristics of this three regions and their combinations.
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 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).
Protein scaffold with N-terminal HisTag and RBS BBa_B0034
NFluc of lactamase fused with dCas9, His tag allows for protein purification
Nlact for N terminal of β-Lactamase
HisTag-GFP_Si4-Tag fusion protein with RBS BBa_B0034
Jasmin Weber, Michelle Zoeller, Marcus Geissler, Stefan Bohn, Simon Linder, Sebastian Barthel
LIP prepro + E. coli ribosomal protein L2 (1-60aa) + YLcwp3 Fusion
This is the cell display fusion protein 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.
In order to replace more convenient, we respectively added BamHI, SalI, NdeI between LIP2 prepro, E. coli ribosomal protein L2 (1-60), GS linker, YLcwp3.
E. coli ribosomal protein L2 was synthesized by IDT. LIP2 prepro and YLcwp3 were cloning from the plasmid JMP62 of our lab.
pSB1C3-6H-F3-dCas9, fusion protein of F3 segment of DHFR fused with dCas9, the His tag facilitates protein purification
The coding sequence of this fusion protein is too long, you'd better amplify is separately.
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.
Protein coding region for the NanoLuc(tm) Luziferase (designed by the Promega Corporation, free for research use), Codon optimized for yeast. The Nanoluc is smaller than other luziferase proteins and yields equal or stronger luminescence readings.
gBlock designed by Team Tuebingen, sequence by Promega
Dispersin B fused with DsbA, with mutation at nucleotide 636 (T to C)
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.
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
Micrococcal Nuclease (DNase) fused at N-terminal with DsbA signal sequence
PAM rich URS J23117 is a promoter made from the existing PAM rich URS J23117 promoter (BBa_K1723001) and mutated. This promoter has PAM (PAM = NGG sequence) rich Upstream Regulatory Sequence to enable the use of protein dCas9-ω (BBa_K1723000) as a gene transcription regulator when in complex with one sgRNA targeting the promoter such as: X0 (BBa_K1723006), X4 (BBa_K1723007), or X35 (BBa_K1723008). Discover all the parts that can work with this one: http://2015.igem.org/Team:EPF_Lausanne/Part_Collection
This part was synthesized on the model of the PAM rich URS J23117 promoter. It was obtained by mutating bases on the promoter to obtain different target sites for sgRNAs for promoter regulation using dCas9-ω system.
Inducible generator of eYFP with C-terminal SH3 ligand
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.
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
Gene for the repressor LsrR, present in the E. coli LsrRK operon. Represses the pLsrA promoter, as well as its own promoter, pLsrR
This part was synthesized from E. coli genomic data retrieved via EcoGene: http://www.ecogene.org/gene/EG13805
mRFP generator under control of T7 promoter
mRFP is synthesized when T7-polymerase is induced.
Both subparts were obtained from iGEM distribution plates.
FP:GTTTCTTCGAATTCGCGGCCGCTTCTAGATTGACTCTGTAGTTGCTACA VP:GTTTCTTCCTGCAGCGGCCGCTACTAGT ATTGCACACC CTGTAGCAAC TACAGA
This is a engineered TAL effector that can bind with the DNA sequence 5'-CTTTCCACAAATAG-3'
Neomycin Phosphotransferase, for a composite biobrick.
UbiX is a flavin prenyltransferase required in ubiquinone synthesis in Escherichia coli.
MerR activator under constitutive promoter and induceble merT promoter with 5 UTR -sfGFP
The covalently bound GPI-anchored cell wall protein of Y. lipolytica Ylcwp1 has been isolated and characterized (Jaafar and Zueco 2004). The nucleotide sequence encoding 110 amino acids of the Ylcwp1 C terminus has been utilized for the construction of the cell surface display vector in Y. lipolytica (Yue et al. 2008). This system was successfully used to immobilize green fluorescent protein (Yue et al. 2008). The ability of five new putative covalently linked cell wall proteins of Y. lipolytica -Ylcwp1, Ylcwp2, Ylcwp3, Ylcwp4, Ylcwp5, was tested and the highest cell-bound lipase value was obtained with the anchor domains Ylcwp3 (Evgeniya et al. 2011), so we utilized the YLcwp3 for the cell surface display in Y. lipolytica.
The source of the part will be its sequence which was retrieved from GenBank, and altered some regions by our lab.
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
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)
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.
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.
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
MaSp2 6 mer in pSB1C3. No regulatory elements present.
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.
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.
zwf - Glucose-6-Phosphate-Dehydrogenase from E.coli
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.
Secretion signal peptide originated from the B.subtilis extracellular protease AprE
pRha - fimH 49 KO - RPMrel - SpyTag_225 - HisTag_225
This is a coding sequence of C. albicans mating pheromone receptor. Cells with this receptor being expressed react to the C. albicans (can be found in the registry as [http://parts.igem.org/Part:BBa_K1690001 BBa_K1690001]) mating pheromone by the activation of the pheromone mating pathway. The coding sequence was codon optimized for the expression in S. cerevisiae. Detailed description can be obtained from the [http://2015.igem.org/Team:Czech_Republic/Project/Synthetic_signals_and_receptors#Receptors iGEM 2015 Czech Republic wiki].
The coding sequence was codon optimized for the expression in S. cerevisiae. Kozak sequence was added before the start codon for a better translation.
tsPurple Reporter + 3-alpha-HSD (RBS + Coding + Terminator)
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
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)
Gene for the C subunit of the LsrACDB transporter, present in E. coli on the LsrACDBFG operon. The C subunit is a permease protein in the transporter, which actively brings autoinducer 2 (AI-2) into the cell.
The portion of the LsrACDBFG operon that codes for the AI-2 transporter consists of four open reading frames, which code for A, C, D, and B, respectively. Three of these ORFs overlap with one another: C overlaps with A, and its RBS is contained within the A subunit ORF, and D overlaps with C (although to a lesser extent), and its RBS is contained within the C subunit ORF. From a modularization standpoint, this creates an inefficiency because the wild type ORFs of the A and C subunits both contain functional RBSs, and in the case of the A subunit ORF, an ATG 4bp downstream. To reduce this inefficiency, we have created versions of each subunit ORF that are optimized for modularization. Further explanation is available in the descriptions of those parts.
This part was synthesized from E. coli genomic data retrieved via EcoGene: http://www.ecogene.org/gene/EG13807
Mcfp-3 is foot protein secreted from Mytilus californianus. The protein is of significance to the formation of byssus to help mussels permanently or temporarily tether to the surface of solid surface of reef or ship-body. This coherent substance shows the excellent adhesion performance with no substitute under water for sustaining the repeating wash of waves and having no toxicity. XPR2 pre is the pre-region 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 (Matoba and Ogrydziak, 1989). The signal tag, XPR2 fused to Mcfp3 can lead the co-translational translocation of heterologous protein Mcfp-3 to be secreted out of the cell to achieve its function, during the progress of which the peptide will be deleted in the Golgi apparatus to eliminate the interference to the secreting protein.
Artilysin Art-175 fused at N-terminal with porin-dependent YebF secretion signal protein
mRFP under control of the chromium responsive promoter
chromium responsive Promoter with RFP for detecition
chromium VI responsive promoter of ????Ochrobactrum tritici???? 5bvl1
LIP2 prepro + E. coli ribosomal protein L2 (203-273aa) + YLcwp3 Fusion
This is the cell display system of Yarrowia lipolytica, composed of LIP2 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, called Si-tag1, Si-tag2, Si-tag3. We respectively test the silica-binding characteristics of this three regions and their combinations.
gRNA for SaCas9 targeting human ubiquitin B gene under U6 promoter
gRNA for SaCas9 targeting ubiquitin B under U6 promoter
fim switch[default OFF](Tokyo_Tech/J23119)
This part codes for a enzymatically inactive Cas9 protein (dCas9) that is fused to rpoZ, the omega subunit of E.Coli RNA Polymerase (RNAP). Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease that targets and cleaves any DNA sequence complementary to its guide RNA (gRNA). Catalytically ???dead??? Cas9 (dCas9) lack the ability to cleave DNA. When fused to a RNAP recruiting element (in this case the omega subunit of RNAP), chimeric dCas9 can act as a programmable transcription activator. dCas9 may also act as a DNA transcription inhibitor: depending on its gRNA-determined binding site, it has been shown in yeasts to sterically hinder RNAP recruitment to promoter sequences.
The original dCas9-ω given to us by the Bikard lab contains an EcoRI restriction site. We performed a site-directed mutagenesis to remove it.
A plasmid containing dCas9_ω (pdCas9) was given to our iGEM team by David Bikard.
This sgRNA sequence was designed to target the Promoter Bba_J23119 with the N20 sequence ttgacagctagctcagtcct . If this gRNA is expressed in a cell with a form of the dCas9 protein, the protein will be targeted to the N20 sequence and repress gene expression. The part is under the control of the promoter Bba_R0040. Promoter Bba_R0040 was chosen because it does not contain a PAM and therefore cannot be inadvertently repressed by dCas9 due to unintentional homology in the N20 sequence. Expression of this part can be repressed if TetR is expressed in the cell without Tetracycline present. If TetR and Tetracycline (or its analog aTc) are both present, the part will be expressed.
We made sure to use a different promoter for this part than the one which the part targets.
The basic template for the sgRNA sequence was taken from the supplementary information of Qi LS, Larson MH, Gilbert LA, et al. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell. 2013;152(5):1173-1183. doi:10.1016/j.cell.2013.02.022.
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].
pSB1C3-6H-dCas9-F3, this is the fusion protein of dCas9-F3, His tag allows for protein purification
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.
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)
MaSp1 9 mer in pSB1C3, no regulatory elements present.
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.
It is the combination of IAAH and termination.
We combine IAAH and the termination to improve the efficiency of the building.
This sgRNA sequence was designed to target the Promoter Bba_R0010 with the N20 sequence atgttgtgtggaattgtgag . If this gRNA is expressed in a cell with a form of the dCas9 protein, the protein will be targeted to the N20 sequence and repress gene expression. The part is under the control of the promoter Bba_R0040. Promoter Bba_R0040 was chosen because it does not contain a PAM and therefore cannot be inadvertently repressed by dCas9 due to unintentional homology in the N20 sequence. Expression of this part can be repressed if TetR is expressed in the cell without Tetracycline present. If TetR and Tetracycline (or its analog aTc) are both present, the part will be expressed.
We made sure to not target the sequence that is promoting the gRNA
Unit for RFC 110 assembly, clonable in RFC 10 vectors like pSB1C3.
Standard assembly construct for RFC 110 assembly
This sgRNA sequence was designed to target the Promoter Bba_J23100 with the N20 sequence ttgacggctagctcagtcct . If this gRNA is expressed in a cell with a form of the dCas9 protein, the protein will be targeted to the N20 sequence and repress gene expression. The part is under the control of the promoter Bba_R0040. Promoter Bba_R0040 was chosen because it does not contain a PAM and therefore cannot be inadvertently repressed by dCas9 due to unintentional homology in the N20 sequence. Expression of this part can be repressed if TetR is expressed in the cell without Tetracycline present. If TetR and Tetracycline (or its analog aTc) are both present, the part will be expressed.
eYFP with C-terminal SH3 ligand and RBS BBa_B0034
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).
LsrR of LuxS/AI-2 signaling pathway in Salmonalla
Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. And autoinducer-2 (AI-2) has been proposed to serve as a 'universal signal' for interspecies communication. In the LuxS/AI-2 signaling system of Salmonella Typhimurium, AI-2 response involves ATP binding cassette transporter encoded by genes named Lsr (LuxS regulated). And LsrR is the Transcriptional regulator which is inactivated by binding phospho-AI-2, leading to the transcription of the lsr genes. In our project, we set this protein-coding part under a nisA promoter and try to integrate them in the genome of Lactobacillus or Lactococcus for the final purpose of constructing an integrated AI-2 response pathway of Salmonella in the engineered bacteria.
The lsrR gene was isolated from the genome of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 using PCR. We added restriction enzyme sites when designing primers, and the assembly is done through the use of restriction sites (cutting and ligating).
We extracted the genomic DNA of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, and used PCR to isolate the gene LsrR(Gene ID: 1255600).
Contains an IPTG inducible promoter an RBS, RFP, and terminator.
gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm under U6 promoter
This gene provides the intermediate for synthesizing gRNA targeting RFP (BBa_J04450). When used in conjuction with the CRISPR/Cas system, this gRNA will bind to and digest the RFP plasmid.
Synthesized by Integrated DNA Technologies (IDT). We amplified the sequence, digested with EcoRI and SpeI and ligated into the PSB1C3 vector.
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
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
Alexandra Goretzki, Tanja Habeck, Nina Kuschik-Maczollek, Laurin Monnheimer, Steven Pilger, Bianca Reisinger, Daniel Stumpf
LIP2 prepro + E. coli ribosomal protein L2 (1-202aa)+ YLcwp3 Fusion
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, called Si-tag1, Si-tag2,Si-tag3. We respectively test the silica-binding characteristics of this three regions and their combinations..
This sgRNA sequence was designed to target the Promoter Bba_R0062 with the N20 sequence tacaggtttacgcaagaaaa . If this gRNA is expressed in a cell with a form of the dCas9 protein, the protein will be targeted to the N20 sequence and repress gene expression. The part is under the control of the promoter Bba_R0040. Promoter Bba_R0040 was chosen because it does not contain a PAM and therefore cannot be inadvertently repressed by dCas9 due to unintentional homology in the N20 sequence. Expression of this part can be repressed if TetR is expressed in the cell without Tetracycline present. If TetR and Tetracycline (or its analog aTc) are both present, the part will be expressed.
Dispersin B with mutation at nucleotide 582 (T to C)
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.
M2-6(1C3) under control of T7 promoter RBS.
Medium constitutive promoter + CsgB & CsgC
CsgB functions as the anchor on the cell wall for curli nanowires. It triggers the aggregation of CsgA and leads to the formation of amyloid nanowires. CsgC is positively involved in the extracellular aggregation of CsgA into amyloid nanowires (curli assembly) in E.coli functioning as a chaperone for CsgA. Constructs with different promoter strenghts were designed to investigate the impact on nanowire self-assembly. Medium expression level.
We inserted non-coding scar sequences after the promoter and RBS to prevent missing initial codons during transcription/translation.
The RBS and CsgC were synthesized from the genomic sequence of E.coli K-12 MG1655. The promoter is part of the constitutive promoter family made by John Anderson from iGEM Berkeley 2006.Non-coding scar sites were designed at random.
This is the translational unit of 3-hexulose-6-phosphate synthase (HPS) from Bacillus methanolicus MGA3. It is codon optimized for E.coli.
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
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.
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.
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
Designed to close lacO present systems in higher temperatures by producing lacI.
The part is a composite part formed up of several distinct parts.
Gene for the D subunit of the LsrACDB transporter, present in E. coli on the LsrACDBFG operon. The D subunit is a permease protein in the transporter, which actively brings autoinducer 2 (AI-2) into the cell. The portion of the LsrACDBFG operon that codes for the AI-2 transporter consists of four open reading frames, which code for A, C, D, and B, respectively. Three of these ORFs overlap with one another: C overlaps with A, and its RBS is contained within the A subunit ORF, and D overlaps with C (although to a lesser extent), and its RBS is contained within the C subunit ORF. From a modularization standpoint, this creates an inefficiency because the wild type ORFs of the A and C subunits both contain functional RBSs, and in the case of the A subunit ORF, an ATG 4bp downstream. To reduce this inefficiency, we have created versions of each subunit ORF that are optimized for modularization.
This version of the open reading frame has been optimized for modularization via silent mutations in segments of code sharing more than 83% homology with the Shine-Dalgarno sequence. It is otherwise identical to the wild type LsrD gene (http://parts.igem.org/Part:BBa_K1799005).
This part was synthesized, and is based on E. coli genomic data retrieved via EcoGene: http://www.ecogene.org/gene/EG13808
RBS found upstream of the LsrA open reading frame in E. coli.
LIP prepro + E. coli ribosomal protein L2 (61-273aa) + YLcwp3 Fusion
This sgRNA sequence was designed to target the Promoter Bba_J23101 with the N20 sequence tttacagctagctcagtcct . If this gRNA is expressed in a cell with a form of the dCas9 protein, the protein will be targeted to the N20 sequence and repress gene expression. The part is under the control of the promoter Bba_R0040. Promoter Bba_R0040 was chosen because it does not contain a PAM and therefore cannot be inadvertently repressed by dCas9 due to unintentional homology in the N20 sequence. Expression of this part can be repressed if TetR is expressed in the cell without Tetracycline present. If TetR and Tetracycline (or its analog aTc) are both present, the part will be expressed.
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.
This is a coding sequence of L. elongisporus mating pheromone together with a secretion tag. Expression in the yeast cell causes secretion of the mature mating pheromone into the medium. Cells with a corresponding STE2 receptor react to this pheromone by the activation of the pheromone mating pathway. Detailed description can be obtained from the [http://2015.igem.org/Team:Czech_Republic/Project/Synthetic_signals_and_receptors#Signals iGEM 2015 Czech Republic wiki].
The secretion tag was obtained by a genomic PCR from S. cerevisiae. The pheromone sequence was synthetized.
This part is derived from [http://parts.igem.org/Part:BBa_K1690000 BBa_K1690000]. Using site-directed mutagenesis, we replaced the original pheromone sequence.
Inducible generator of Humicola insolens cutinase (HIC) with N-terminal His-Tag
Sebastian Barthel, Steven Pilger, Jonas Sindlinger, Bianca Reisinger, Sven Jager
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.
Gene for the B subunit of the LsrACDB transporter, present in E. coli on the LsrACDBFG operon. The B subunit is the autoinducer 2 (AI-2) binding protein in the transporter, which actively brings AI-2 into the cell. The portion of the LsrACDBFG operon that codes for the AI-2 transporter consists of four open reading frames, which code for A, C, D, and B, respectively. Three of these ORFs overlap with one another: C overlaps with A, and its RBS is contained within the A subunit ORF, and D overlaps with C (although to a lesser extent), and its RBS is contained within the C subunit ORF. From a modularization standpoint, this creates an inefficiency because the wild type ORFs of the A and C subunits both contain functional RBSs, and in the case of the A subunit ORF, an ATG 4bp downstream. To reduce this inefficiency, we have created versions of each subunit ORF that are optimized for modularization.
This version of the open reading frame has been optimized for modularization via silent mutations in segments of code sharing more than 83% homology with the Shine-Dalgarno sequence. It is otherwise identical to the wild type LsrB gene (http://parts.igem.org/Part:BBa_K1799006).
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.
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.
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.
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.
This is the translational unit of the 6-phospho-3-hexuloisomerase (PHI) from Bacillus methanolicus MGA3. It is codon optimized for E.coli.
Mercury responsive promoter with UTR-sfGFP
mercury responsive promoter with 5??untranslated region and sfGFP for detection
Chromium responsive promoter with UTR+RBS and sfGFP
Chromium responsive promoter with 5?? untremslated region and sfGFP for detection
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
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
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.
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.
Ferulic acid decarboxylase (FDC) with T7 promoter and RBS
Site directed mutagenesis of SpeI restriction enzyme site within sequence.
We choose a strong promoter+ strong RBS to construct L-Lactate part. The NAD+-dependent L-lactate dehydrogenase gene is from Lactobacillus casei.
This sgRNA sequence features a scrambled N20 sequence designed to serve as a negative control for dCas9 gene repression studies. The part is under the control of the promoter Bba_R0040. Promoter Bba_R0040 was chosen because it does not contain a PAM and therefore cannot be inadvertently repressed by dCas9 due to unintentional homology in the N20 sequence. Expression of this part can be repressed if TetR is expressed in the cell without Tetracycline present. If TetR and Tetracycline (or its analog aTc) are both present, the part will be expressed.
The N20 sequence was used with Origene???s permission and was taken from Origene???s pCas Scramble (GE100003). The basic template for the sgRNA sequence was taken from the supplementary information of Qi LS, Larson MH, Gilbert LA, et al. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell. 2013;152(5):1173-1183. doi:10.1016/j.cell.2013.02.022.
ptrp is a promoter with the trp operator. It will be repressed by trpR (K588000) and LovTAP. Note that the original trp operator contains two SpeI sites。 We remove the illegal sites without affecting its function by site-directed mutagenesis for meeting the requirements of RFC10 and more convenient to be assembled, and finally constructed three ptrp muant, called ptrp mutant1, ptrp mutant2, and ptrp mutant3.
Cloned from BBa_K588001 and mutated by overlap extension PCR.
Functional nitrate anion sensor based on the NarX/NarL Two-Component Signal Transduction System in E. coli with semi-tunable dynamic range and sensitivity. NarL expression is regulated by the TetR-PLTet0-1 1-component regulatory system, whereby addition of ATC (anhydrotetracycline) induces higher NarL expression. This regulates the ratio of native NarX/NarL protein in the cell, controlling to an extent the dynamic range and sensitivities of the sensor. Phosphorylated NarL binds to the repressible PdcuS promoter from E. coli, regulating the expression of sfGFP. Low nitrate conditions result in high sfGFP expression, high nitrate conditions repress transcription. The sensitivity of the sensor is in the sub-mM to mM range for nitrate. Further testing is needed to fully characterize the relationship of ATC-induction to nitrate sensitivity and dynamic range. The sensor is functional in the absence of ATC with a low dynamic range, and ATC concentrations of 1-10 ng/ml increase dynamic range and sensitivity. Further increase of ATC drives the ON-state down, decreasing dynamic range.
The PdcuS promoter was chosen for its NarL binding site, repressible activity, and the absence of other protein-binding domains, suggesting a robust, orthogonal response. The construct was assembled using Golden Gate Assembly.
NarL, PdcuS - E. coli MG1655 genome sfGFP,TetR-PLTet0-1 scaffold - pRS334 (courtesy of Tabor Lab, Rice Univeristy)
Cis-aconitate decarboxylase (cadA) from Aspergillus terreus
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
pSB1C3-6His-F[1,2]-dCas9, segment F[1,2] fusion with dCas9, 6His tag allows for protein purification.
Gene for the C subunit of the LsrACDB transporter, present in E. coli on the LsrACDBFG operon. The C subunit is a permease protein in the transporter, which actively brings autoinducer 2 (AI-2) into the cell. The portion of the LsrACDBFG operon that codes for the AI-2 transporter consists of four open reading frames, which code for A, C, D, and B, respectively. Three of these ORFs overlap with one another: C overlaps with A, and its RBS is contained within the A subunit ORF, and D overlaps with C (although to a lesser extent), and its RBS is contained within the C subunit ORF. From a modularization standpoint, this creates an inefficiency because the wild type ORFs of the A and C subunits both contain functional RBSs, and in the case of the A subunit ORF, an ATG 4bp downstream. To reduce this inefficiency, we have created versions of each subunit ORF that are optimized for modularization.
This version of the open reading frame has been optimized for modularization via silent mutations of the LsrD RBS, as well as any segments of code sharing more than 83% homology with the Shine-Dalgarno sequence. It is otherwise identical to the wild type LsrC gene (http://parts.igem.org/Part:BBa_K1799004).
This part was synthesized, and is based on E. coli genomic data retrieved via EcoGene: http://www.ecogene.org/gene/EG13807
Chromium responsive promoter under T7-promoter with UTR-sfGFP
T7 controlled Chromium responsive promoter with 5??untranslated region and sfGFP for detection
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
Sequence for pTet inverting regulator driven by the TetR protein.
BBa_R0040 TetR-Regulated Promoter is based on a cI promoter. It has been modified to include two TetR binding sites and the BioBrick standard assembly head and tail restriction sites.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210.
LIP prepro + E. coli ribosomal protein L2 (61-202aa) + YLcwp3 Fusion
This is the cell display system of Yarrowia lipolytica, composed of LIP2 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, called Si-tag1, Si-tag2, Si-tag3. We respectively test the silica-binding characteristics of this three regions and their combinations.
In order to replace the domains more conveniently, we added BamHI, SalI, NdeI, NdeI between LIP2 prepro, E. coli ribosomal protein L2, GS linker and YLcwp3.
MaSp1 and MaSp2 12-mer block co-polymer under control of T7 promoter and RBS. Ratio of 2 MaSp1 to 1 MaSp2.
T7 promoter for expression of functional RNA
pAga1: mating factor alpha responsive promoter
pAga1 is a mating factor alpha responsive promoter that is found endogenously in MATa S. Cerevisiae (budding yeast). pAga1 activates the Aga1 gene, which contributes in the S. Cerevisiae mating pathway by supporting cell structure as mating yeast cells project, or "shmoo" towards each other. pAga1 is activated by the presence of mating factor alpha (aka alpha factor) which is endogenously secreted by MATα type cells.
pAga1 is found in the genomic sequence of MATa S. Cerevisiae.
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.
The hepatitis delta virus ribozyme shows a specific and highly efficient cleavage activity at the RNAs 5' end of the sequence. Therefore it is highly suitable to create defined ends for functional RNA expression.
Cleavage occurs upstream of the G on the 5' end.
Optimized sequence derived from hepatitis delta virus genome.
MaSp1 and MaSp2 12-mer block co-polymer under control of T7 promoter and RBS. Ratio of 1 MaSp1 to 1 MaSp2.
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.
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.
fim switch[default OFF](wild-type)_rbs_gfp
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)
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.
MaSp1 and MaSp2 12-mer block co-polymer in pSB1C3, no regulatory elements. Ratio of 2 MaSp1 to 1 MaSp2.
gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm
LsrK of LuxS/AI-2 signaling pathway in Salmonalla
Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. And autoinducer-2 (AI-2) has been proposed to serve as a 'universal signal' for interspecies communication. In the LuxS/AI-2 signaling system of Salmonella Typhimurium, AI-2 response involves ATP binding cassette transporter encoded by genes named Lsr (LuxS regulated). And LsrK is the kinase which catalyzes the phosphorylation of autoinducer 2 (AI-2) to phospho-AI-2. In our project, we set this protein-coding part under a nisA promoter and try to integrate them in the genome of Lactobacillus or Lactococcus for the final purpose of constructing an integrated AI-2 response pathway of Salmonella in the engineered bacteria.
The lsrK gene was isolated from the genome of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 using PCR. We added restriction enzyme sites when designing primers, and the assembly is done through the use of restriction sites (cutting and ligating).
We extracted the genomic DNA of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, and used PCR to isolate the gene LsrK(Gene ID: 1255599).
codon optimized PAL with T7 promoter and Flag Tag
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.
fim switch[default ON](Tokyo_Tech/R0010)
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.
RBS located upstream of the LsrC open reading frame in E. coli
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)
LsrD of LuxS/AI-2 signaling pathway in Salmonalla
Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. And autoinducer-2 (AI-2) has been proposed to serve as a 'universal signal' for interspecies communication. In the LuxS/AI-2 signaling system of Salmonella Typhimurium, AI-2 response involves ATP binding cassette transporter encoded by genes named Lsr (LuxS regulated). And LsrD is thetransmembrane protein which constructed the ABC transport. In our project, we set this protein-coding part under a nisA promoter and try to integrate them in the genome of Lactobacillus or Lactococcus for the final purpose of constructing an integrated AI-2 response pathway of Salmonella in the engineered bacteria.
The lsrD gene was isolated from the genome of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 using PCR. We added restriction enzyme sites when designing primers, and the assembly is done through the use of restriction sites (cutting and ligating).
We extracted the genomic DNA of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, and used PCR to isolate the gene LsrA(Gene ID: 1255603).
RBS located upstream of the LsrD open reading frame (inside the LsrC open reading frame) in E. coli
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
This is the translational unit for the phosphoketolase (XPK). It derives from Bifidobacterium adolescentis and is codon optimized for E. coli.
The recombinant promotor hp4d, nearly unaffected by pH, carbon source, nitrogen source and some conditions, can strongly promote gene expression in any culture medium(Madzak et al., 2000). Promoter hp4d is composed of four tandem repeated UAS1 sequences in upstream and the small LEU2 promoter, which is the sequence between TATA box and start condon ATG of LEU2 promoter in downstream. UAS1 is the upsteam activating sequence of the pXPR2, which is less affected by other conditions. Though any inducers are non-essential for activiation of php4d during cultivating, hp4d, activated by unknown element, isn't constitutive promoter. The gene promoted by promoter hp4d usually expresses at the early stage of stabilization, called growth phase dependent (Nicaud et al., 2002). This feature, separating the strain growth and producing foreign protein, is useful for production of foreign protein, which avoids the harm by expressing foreign protein.
MaSp1 and MaSp2 12-mer block co-polymer in pSB1C3. No regulatory elements present. Ratio of 1 MaSp1 to 1 MaSp2.
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.
Gene for the B subunit of the LsrACDB transporter, present in E. coli on the LsrACDBFG operon. The B subunit is the autoinducer 2 (AI-2) binding protein in the transporter, which actively brings AI-2 into the cell.
This part was synthesized from E. coli genomic data retrieved via EcoGene: http://www.ecogene.org/gene/EG13809
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
Mcfp-3 is foot protein secreted from Mytilus californianus. The protein is of significance to the formation of byssus to help mussels permanently or temporarily tether to the surface of solid surface of reef or ship-body. This coherent substance shows the excellent adhesion performance with no substitute under water for sustaining the repeating wash of waves and having no toxicity. The signal tag, LIP2 prepro(BBa_K1592000), fused to Mcfp3 can lead the co-translational translocation of heterologous protein Mcfp-3 to be secreted out of the cell to achieve its function, during the progress of which the peptide will be deleted in the Golgi apparatus to eliminate the interference to the secreting protein. And hexahis-tag added between LIP2 prepro and Mcfp3 can therefore be used for protein purification.
pRha - fimH 136 KO - RPMrel - SpyTag_225 - HisTag_225
gRNA for SaCas9 targeting human ubiquitin B gene
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.
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.
Coding device for anti-microbial peptide derived from Methyloversatilis universalis EcnB.
Provides Kanamycin Resistance to the cell under the Promoter R0010.
Copper responsive promoter with T7-promoter and UTR-sfGFP
This part consists of the human antichymotrypsin with a terminator attached behind it.
This part comes from a human cDNA clone attached with the iGEM terminator B0015.
eYFP with C-terminal GBD ligand and RBS BBa_B0034
HisTag-Scaffold_Si4-Tag fusion protein with RBS BBa_B0034
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
LsrA of LuxS/AI-2 signaling pathway in Salmonalla
In a process called quorum sensing, bacteria communicate with one another using secreted chemical signaling molecules termed autoinducers. As the density of the bacterial population increases, so does the concentration of secreted autoinducer molecules. When the concentration of the autoinducer reaches a critical threshold, it diffuses back into the cell and activates or represses certain target genes. An autoinducer called AI-2 is generated by many species of Gram-negative and Gram-positive bacteria. In the LuxS/AI-2 signaling system of Salmonella enterica serovar Typhimurium, AI-2 response involves genes named lsr (LuxS regulated) that encode ATP binding cassette-type transporter. And LsrA is ATP-binding protein of the ABC transporter. In our project, we set this part under a nisA promoter for the final purpose of constructing an integrated AI-2 response pathway in our engineering bacteria.
The lsrA gene was isolated from the genome of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 using PCR.We added restriction enzyme sites when designing primers,and the assembly is done through the use of restriction sites (cutting and ligating).
It comes from the genome of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2(Gene ID: 1255601)
MaSp2 3-mer in a T7-RBS expression vector (BBa_K552998)
We incorporated tag1 into a RFP-based test platform for inducible protein degradation in E. coli.
In the presence of AHL, produces LuxR, which generates a positive feedback loop increasing transcription. LacZ is located downstream of LuxR and is used to measure promoter activity through a β-galactosidase assay.
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.
MaSp1 9 mer under control of T7 promoter and RBS
Michael Osthege, Sarah Lubjuhn, Laura Helleckes
This is the translational unit of GlgC. It creates ADP-glucose from glucose-1-phosphate for glycogen synthesis.
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.
fim switch[deault ON](Tokyo_Tech/J23119)
Promoter for the E. coli LsrACDB operon. This promoter is repressed by LsrR, and derepressed by autoinducer 2 (AI-2) in the presence of LsrK.
Promoter begins with the start of its cAMP-CRP binding site and ends after the second key LsrR binding sequence.
This part was synthesized from E. coli genomic data retrieved via EcoGene. It is based on, but not identical to, a part created by the 2011 Tokyo Tech iGEM team: http://parts.igem.org/Part:BBa_K649100
Major Spidroin Protein 1 (MaSp1) with Sticky Ends AB
pAra + fusion protein BFP_Spycatcher_His
Inducible arabinose promoter controlling expression of BFP_SpyCatcher_His.
The start condon of SpyCatcher and the stop codon of BFP needed to be removed to ensure that one complete protein was expressed.
These part was synthesized as linear DNA and only restriction/ligation was done to insert this into the backbone.