This part has two fused proteins downstream of an arabinose-inducible promoter: CpxR-IFP[1] and CpxR-IFP[2]. This construct was made in all orientations: - CpxR-IFP[1] + CpxR-IFP[2] (BBa_K1286008) - IFP[1]-CpxR + CpxR-IFP[2] (BBa_K1486010) - CpxR-IFP[1] + IFP[2]-CpxR (BBa_K1486011)
All orientations were made, so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used for each of the fused proteins, and an RBS was put before each one of them.
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.
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.
Calgary's BBa_K339007 repaired (part didn't have the sequence they said they had.
Theophylline Responsive Riboswitch 1G1 with Engineered RBS+GFP generator
Siyang HAO, Tong MU, Zheng LUO, Mengqi ZHANG
This is a GFP generator with a theophylline responsive riboswitch sequence. The mRNA with a theophylline riboswitch in it would respond to theophylline concerntration, producing different amount of GFP. Under the control of different promoters, it could be used to demonstrate the theophylline responsive curve, providing data for modeling. The theophylline riboswitch sequence could also be used to regulate the translation of other genes.
The RBS of this device is altered into AGGAGGU. When changing the RBS, the anti-RBS sequence should be changed as well.
Theophylline riboswich is composed of a synthesized theophylline aptamer and a regulatory platform.
Theophylline responsive riboswitch Theo 1G1
CpxR is a transciption factor that dimerizes and becomes active when phosphorylated by the transmembrane histidine kinase CpxA. CpxA in turn becomes active when the bacterial cell is submitted to different kinds of shock (pH, temperature, pressure, protein misfolding in the inter-membrane space...).
When making composite parts with CpxR that are supposed to dimerize, we had to take care about the positioning of the other elements regarding the parallel dimerization of CpxR.
The part has been extracted from the E coli genome.
Mhbpt: a promoter that can be induced by 3-HBA and MhbR.
Mhbpt is a promoter that can be induced by the compound of MhbR and the 3-HBA. The promoter contains the binding site to bind the complex. It is a bidirectional promoter for the two promoters have a partial overlap.
The gene can be gotton from the bacteria M5a1, and the sequence comes from GenBank https://www.ncbi.nlm.nih.gov/nuccore/EU165545.1
Transcription Factor Binding Site from Cyanobacterial Pcpc560
A sensor that activates in the presence of nitrate in anaerobic conditions in E. coli.
This part will used along with a Green fluorescent actuator to test for the presence of nitrate.
We had to deal with finding the appropriate binding areas for the promote without including any of the sequence that codes for proteins.
The source for this part comes from the NIR promoter region in E. coli plasmid.
This part is composed of an arabinose-inducible promoter for superfolded GFP attached by a linker to CpxR (on the C terminus of CpxR).
We had to test both attachments, N and C terminus of the CpxR.
The arabinose promoter and the sfGFP were obtained from the registry, while the CpxR was obtained from the E coli genome.
This part is composed of an arabinose-inducible promoter for superfolded GFP attached by a linker to CpxR (on the N terminus of CpxR).
This part has a fused protein downstream of an arabinose-inducible promoter: CpxR-IFP[1]. Biobrick BBa_K1486013 has CpxR-IFP[2] with an arabinose promoter. This construct was made in both orientations: BBa_K1486014 is IFP[1]-CpxR (with an arabinose promoter).
Both orientations were made, as well as for IFP[2] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put before each one of them.
This is the DNA sequence of a Y-type hammerhead ribozyme that targets and cleaves the PenI repressor (Part BBa_C0074). This ribozyme has target-binding flanking arms adjacent to the Y-type hammerhead catalytic core and synthetic extended arms located towards the 5' and 3' ends. The catalytic core allows for the sequence-specific cleavage of sites within the coding sequence of PenI. Hammerhead ribozymes are able to cleave at any NUH within an RNA sequence, where H represent any nucleotide except guanine (Mir 2001), although with GUC as the hammerhead consensus cleavage site (Lieber, 1995). The binding of the flanking arms adjacent to the hammerhead catalytic core favours the recognition and cleavage of only one target site. Additional secondary structure within the flanking arms was detected to a small degree by using IDT OligoAnalyzer's Hairpin tool (Owczarzy 2008) as is depicted below. [image] The synthetic extended arms are designed to bind to the complementary extended arms of another similar PenI-cleaving ribozyme (Part _____), but do not contribute to any additional secondary structure as confirmed via the Hairpin tool in IDT's OligoAnalyzer (Owczarzy 2008), as both arms of a particular ribozyme only contain a purine and pyrimidine pair that do not bind to each other (in this case, A and G). The two-nucleotide extended arms are also staggered in number to avoid slippage in binding and bind to its complement pair at significantly higher melting temperatures than the adjacent flanking arms. This feature allows for the sequestering of the two ribozymes when both are present and significantly decreasing any ribozyme activity. Regions of complementarity between the two target-binding regions of both ribozymes are also present to ensure that the functional catalytic cores are not able to cleave their mRNA targets, even when bound. The characteristics of this expressed pair of ribozymes were considered to create a functional RNA-based XOR logic gate. References Mir, A. A., Lockett, T. J. & Hendry, P. Identifying ribozyme-accessible?? sites using NUH triplet-targeting gapmers. Nucleic Acids Res 29, 1906???1914 (2001). Lieber, A. & Strauss, M. Selection of efficient cleavage sites in target RNAs by using a ribozyme expression library. Mol. Cell. Biol. 15, 540???551 (1995). Owczarzy, R. et al. IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res 36, W163???W169 (2008).
Although this particular part is likely to only cleave the PenI repressor mRNA due to its sequence specificity, the simple in silico design of this ribozyme allows for the cleavage of virtually any RNA sequence containing a cleave site (NUH). This particular sequence was obtained by first scanning our target PenI mRNA for the optimal cut site GUC. The 10 nucleotides surrounding this cut site were obtained and the complement of this area was generated for the ribozyme flanking arms. The length of this region was decided by ideal melting temperature (51.4 degrees Celsius). Additional synthetic extended arms were subsequently added to the 5' and 3' ends of the sequence for binding to the complement ribozyme in an XOR gate. This region of non-pairing purines and pyrimidines was staggered in base repeats to avoid slippage of binding and was defined in length by significantly higher melting temperatures (approximately 68 degrees Celsius). Finally, the catalytic core of the Y-type hammerhead ribozyme was introduced at the appropriate region. Throughout this entire process, secondary was continually checked and minimized using IDT OligoAnalyzer's Hairpin tool (Owczarzy 2008). The complete ribozyme sequences was also BLASTed again the E.coli genome, which was our chassis and yielded insignificant results. Other factors such as differential length or target binding regions due to varying melting temperature and GC content were also considered during the in silico design of this ribozyme and its pair in the XOR logic gate. References Owczarzy, R. et al. IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res 36, W163???W169 (2008).
The target-binding flanking arms of the ribozyme were obtained from the complement of the PenI Repressor sequence.
To edit. Pense ?? rajouter les linkers, je ne sais pas s'ils sont de igem ou s'il faut ajouter la s??quence ?? la registry. Check pour tous tes biobricks et dis-moi, please.
This part has the arabinose regulated promoter (AraC/pBAD) upstream of a ribosome binding site. This is ideal for inserting a protein to be expressed downstream of this part as it's expression can be regulated by the presence of the concentration of arabinose in the growing environment/media of the cells. ==What this part actually does== This composite part includes the AraC/pBAD promoter and a strong ribosome binding site designed from the Anderson family of RBS. The AraC/pBAD promoter can be used with prokaryotic chassis to regulate expresion of proteins. When the AraC/pBAD promoter is placed upstream of your canditate translational unit (protein to be expressed) it allows control of the trancription of the said protein. This is done via controlling the concentration of arabinose sugar in the environment/solution of bacterial growth. Controlling the transcription will not ensure translation. The ribosome binding site is ideally placed a small distance away from the promoter site to ensure that the ribosome will bind and start translating the protein coding sequence found downstream.
The part was designed with RFC10 assembly from supplied biobricks of the iGEM2009 distribution.
The AraC promoter can be found naturally at 1.52min of the E.coli genome ([http://ecogene.org/geneinfo.php?eg_id=EG10054 EcoGene number EG10054]. The RBS was taken from the designed sequences of RBS by the Anderson family. For more information visit the original part page [http://partsregistry.org/Part:BBa_J61100 here].
The part contains CpxR with an upstream arabinose-inducible promoter.
The arabinose had to be upstream of the CpxR. Also, the sequence had to be tested because of a mutation, and it works perfectly.
The CpxR comes from the genome of E coli, and the arabinose promoter comes from a biobrick.
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.
The AraC/pBAD promoter can be used with prokaryotic chassis to regulate expresion of proteins. When the AraC/pBAD promoter is placed upstream of your canditate translational unit (protein to be expressed) it allows control of the expression of the said protein. This is done via controlling the concentration of arabinose sugar in the environment/solution of bacterial growth. This composite part does not have an LVA degradation tag at its end, hence can be used with all prokaryotic assemblies.
The part was designed and assembled from the iGEM2009 kit with standard RFC10 assembly.
The AraC promoter is found naturally in E.Coli at 1.52 min/centisome in the clockwise orientation. More information can be found at [http://ecogene.org/geneinfo.php?eg_id=EG10054 EcoGene].
The ferric uptake repressor of Bacillus subtilis called YqkL is a regulator that when in contact with iron will bind and repress a regulatory sequence (the Fur box).
We sequenced the Fur box. This can be added in front of genes to provide regulation when in contact with iron.
This part is the twin of the non-compatible biobrick BBa_K1486008 rendered compatible by mutating the two illegal restriction sites. However, all experiments were run on the aforementioned biobrick.
Site-directed mutagenesis was used to remove the restriction sites in the sequence.
http://www.addgene.org/browse/article/8177/ which was then mutated by us.
This part is for the characterisation of the Invertible promoter BBa_K1768001.
BBa_K1768001 BBa_B0034 BBa_E1010 This part was synthesised by IDT
Modified constitutive promoter, which can be repressed by TetR and induce by tetracycline or aTc.
Parts J23100 through J23119 are a family of constitutive promoter parts isolated from a small combinatorial library. B0030 is a strong RBS based on Ron Weiss thesis.
This part has a fused protein downstream of an arabinose-inducible promoter: IFP[1]-CpxR. Biobrick BBa_K1486015 has IFP[2]-CpxR with an arabinose promoter. This construct was made in both orientations: BBa_K1486012 is CpxR-IFP[1] (with an arabinose promoter).
Both orientations were made, as well as for IFP[2] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put between the promoter and the cds.
This sequence comes from RhodococcusjostiiRHA1. Rhodococcus owns the ability of degrading terephthalic acid (TPA), this pathway is regulated by many genes and some of them can be induced by TPA. The inducing effect is based on a regulatory proteintpaR, which can bind this sequence at the existence of TPA and induce the expression of downstream gene. This sequence should be placed before the sequence of promoter.
This sequence is very short so if the gel recycling is necessary, the agarose concentration should be increased.
It comes from the genome of RhodococcusjostiiRHA1.
This biobrick contains the BBa_R0011 promoter of 80 bp cloned in pSB1C3 with EcoRI and XbaI restriction site which will can be induced by IPTG. RBS is added to the end of the sequence in order to enable gene expression to perform well.
AHL regulated PoPS generator: Las receiver device
Quorum sensing is the mechanism of communication between bacteria via synthesizing and detecting chemical compounds in the media. With these mediators, the promoters and their yield can be tuned or regulated. This part represents an alternative quorum sensing receiver device, which is able to generate a PoPS response in the presence of 6HSLO3 produced by LasI, instead of BBa_F2620. This will allow iGEMers to establish communications between different bacteria colonies and it makes simple to generate easy regulated complex systems. This part is assembled with Las promoter system to improve the current quorum sensing devices and parts which commonly base on Lux promoter system.
Note that pLas has no RBS afterwards, the assembling methods must be selected after this consideration.
pLas and LasR sequences are gathered from Registry.
PMcl1, hemolymph inducible promoter from Metarhizium anisopliae
The ubiquitous fungal pathogen Metarhizium anisopliae kills a wide range of insects. Host hemocytes can recognize and ingest its conidia, but this capacity is lost on production of hyphal bodies. The unusual ability of hyphal bodies to avoid detection depends on a gene (Mcl1) that is expressed within 20 min of the pathogen contacting hemolymph. Mcl1 (for Metarhizium collagen-like protein)
It was isolated from the genomic DNA of our chassis organism Metarhizium anisopliae ARSEF549.
A part used to quantify the AraC-RBS construct <partinfo>K259007</partinfo> created by the BCCS-Bristol iGEM'09 team.
This part was designed with the specified biobricks from the parts registry. AraC occurs naturally in E.coli. GFP occurs in jellyfish species, RBS designed by the Anderson family and Terminator by Resma Shetty.
MhbRpt is a complex promoter which can be induced by 3-HBA.
Besides the MhbR, there is a promoter named Mhbpt which can be induced by the compound of MhbR and the 3-HBA. For there has been the MhbR, only the 3-HBA will induce the complex promoter. It can be used as an induced start system.
One bp of G was mutated to the A to avoid the formation of the PstⅠ.
The gene can be gotton from the bacteria M5a1, and the sequence comes from GenBank
This part has a fused protein downstream of an arabinose-inducible promoter: IFP[2]-CpxR. Biobrick BBa_K1486014 has IFP[1]-CpxR with an arabinose promoter. This construct was made in both orientations: BBa_K1486013 is CpxR-IFP[2] (with an arabinose promoter).
Both orientations were made, as well as for IFP[1] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put before each one of them.
Quorum sensing is the mechanism of communication between bacteria via synthesizing and detecting chemical compounds in the media. With these mediators, the promoters and their yield can be tuned or regulated. This part represents an alternative quorum sensing sender device which is designed to induce PoPS production in the parts including pLas such as BBa_K772004. LasI is an enzyme generator gene sequences which synthesizes 6HSLO3 (AHL) in order to induce a PoPS response in the pLas promoter. pLas promoter works in the presence of both 6HSLO3 and LasR protein.
Note that there is an RBS sequence just before LasI; in order to be easily used with quorum sensing receiver devices such as BBa_F2620 or BBa_K772004.
LasI sequence is gathered from Registry.
This part has a fused protein downstream of an arabinose-inducible promoter: CpxR-IFP[2]. Biobrick BBa_K1486012 has CpxR-IFP[1] with an arabinose promoter. This construct was made in both orientations: BBa_K1486015 is IFP[2]-CpxR (with an arabinose promoter).
IPTG/Lactose inducible expression casette for B. Subtilis
This part in an IPTG/Lactose inducible expression casette for Bacillus Subtilis. It is build to have a slow and controlable response with the quantity of IPTG introduced in the medium.
The experiments have still to be done. The host plasmid is pSB1K3
This part is the fusion of the biobrick K606010 and K143055
This promoter can be induced by IPTG. Wild type LacI binding site is replaced with a perfect palindromic "oid" operator sequence which increases the repression by LacI. This biobrick is an enhancement of Biobrick BBa_K090501 which has the sequence from pmutin2.
pmutin2's genbank accession number is AF072806. Although there is no sequence information for pmutin4, it can be derived from pmutin2's sequence information by changing the O1 lacI binding site with the "oid" sequence.
This sequence is taken from pmutin4 integration vector. pmutin2's lacI binding site was changed with the "oid" sequence to create pmutin4.
This part has two fused proteins downstream of an arabinose-inducible promoter: CpxR-IFP[1] and CpxR-IFP[2]. This construct was made in all orientations: - IFP[1]-CpxR + IFP[2]-CpxR (BBa_K1286009) - IFP[1]-CpxR + CpxR-IFP[2] (BBa_K1486010) - CpxR-IFP[1] + IFP[2]-CpxR (BBa_K1486011)