Transcriptional Regulator and cioAB expression platform
The transcriptional regulator come from Pseudomonas pseudoalcaligenes, the cioAB gene complex come from Pseudomonas aeruginosa and the other genes are well characterized biobricks from the registry.
This is the expression platform for our brand new biobricks. We want both the cioAB gene complex to express constitutively so the bacteria, in this case E. coli, can resist to diferent levels of cyanide. We also want the transcriptional regulator to be constitutive because it will interact with the cyanide to further activate activate the inducible promoter
We are working very hard with this construct for further characterization.
This biobrick is product of a ligation from part BBa_K944006 and BBa_K944008
CioAB genes code for a cyanide-insensitive terminal oxidase that gives bacteria another pathway for the electrons transport. In this part, it is coupled with a reporter gene (GFP) and a terminator.
T7 constitutive promoter with RBS, transcriptional regulator from de cyanide inducible promoter and
Most of the parts of this intermediate construct are well characterized biobricks from the registry except for the transcriptional regulator which is a brand new biobrick we add to the registry.
This BioBrick is the ligation of the biobrick BBa_K944009 and the biobrick BBa_K944010 which is an intermediate part to form our composite part which aims to express the cioAB cyanide-insensitive terminal oxidase related to the cytochrome bd quinol oxidases.
This is the ligation of two existing biobricks from the registry.
For more information about this two parts please visit the page of the BioBricks BBa_E0040 for GFP and BBa_B0015 for the double terminator.
For more information about this two parts please visit the page of the BioBricks BBa_I719005 for T7 promoter and BBa_B0034 for the RBS.
CioAB genes - Cyanide-Insensitive terminal oxidase
This biobrick is product of a ligation from CioA and CioB.
CioAB has an important role in allowing aerobic respiration under cyanogenic growth conditions, because it encodes the subunits of a terminal oxidase homologous to the cytochrome bd quinol oxidases in bacteria. Under low oxygen tensions, these proteins provide an alternative to electrons transport. We used this part to give resistance to cyanide to our bacteria under low oxygen tension.
Transcriptional regulator of the cyanide inducible promoter and RBS
This is the ligation of the Transcriptional Regulator from the cyanase operon of Pseudomonas pseudoalcaligenes CECT5344 existing biobricks from the registry.
bbThis BioBrick is the ligation of our brand new Transcriptional Regulator (BBa_K944002) and the RBS (BBa_B0034) which is an existing biobrick from the registry.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
Double terminator consisting of BBa_B0010 and BBa_B0012
Transcriptional Regulator for Cyanide Inducible Promoter
This part came from the genomic sequence of Pseudomonas pseudoalcaligenes CECT5344 and was synthesized by IDT technologies.
The cynF gene codes for a sigma ?54-dependent transcriptional regulator of 648-amino-acid residues, which belongs to the FIS (factor for inversion stimulation) family of regulatory proteins. P. pseudoalcaligenes CynF shared 59% identity with R. palustris CynF (YP_485671) and 58% identity with Bradyrhizobium japonicum CynF (NP_772375). This Transcriptional regulator works along with the cyanide and cyanide compounds inducible promoter in the natural ocurring cyanase operon from P. pseudoalcaligenes.
This part had 3 illegal Pst1 restriction sites.
cioA gene related to cyanide insensitivity
This came from the Pseudomonas aeruginosa genomic sequence we searched in genbank and synthesized by IDT Technologies.
cioA encodes a hydrophobic protein of 488 amino acid residues with a predicted molecular mass of 54241 Da.cioA form part of the cioAB operon from Pseudomonas aeruginosa which code for a novel cyanide-insensitive terminal oxidase related to the cytochrome bd quinol oxidases.
This part has no restriction sites used in the protocol standard 10.
cioB gene related to cyanide insensitivity
This part came from the Pseudomonas aeruginosa genomic sequence and synthesized by IDT Technologies.
cioB encodes a protein, which has the characteristics of a membrane protein, containing 335 amino acids with a predicted molecular mass of 3706 Da. cioB form part of the cioAB operon from Pseudomonas aeruginosa which code for a novel cyanide-insensitive terminal oxidase related to the cytochrome bd quinol oxidases.
The cioB naturaly had a Pst1 restriction site in its sequence so we did a silent mutation for no longer to be this restriction site and proceed with the standard protocol 10.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Transcriptional terminator consisting of a 64 bp stem-loop.
Conversion of the iGEM parts registry to SBOL2.1