The part was chemically synthesised. The sequences were based on parts available in Registry.
This part encodes two enzymes, rhlA and rhlB (BBa_K653000), for biosynthesis of rhamnolipid. The enzymes are under T7 promoter with medium strengh and a strong ribosomal binding site (BBa_K613010), and stopped with a double terminator (BBa_B0015). The promoter had the second highest efficiency as shown by EPFL team 2011 (http://parts.igem.org/Part:BBa_K613010:Experience).
We include the scar sequences between RBS-coding and coding-terminator in hope that it will function properly.
Double terminator consisting of BBa_B0010 and BBa_B0012
T7 promoter family member (LacI repressed)
Parts K613007 through K613012 are T7 promoter variants containing the lac operator just downstream of the T7 promoter. K613007 contains the T7 promoter wildtype sequence. The other sequences were derived by single base pair mutations that have been shown to alter its strength. The part includes a strong ribosomal binding site. Its strength was determined by cloning it in front of mRFP1 reporter and measuring fluorescence of BL21 cells containing a low copy number plasmid containing the reporter construct.
Imburgio, D., Rong, M., Ma, K. & McAllister, W.T. Studies of promoter recognition and start site selection by T7 RNA polymerase using a comprehensive collection of promoter variants. Biochemistry 39, 10419???10430 (2000).
Alessandro Ferrari, Vincent Zimmern, Nadine Guenat, Henrike Niederholtmeyer
Re-designing "The Biosurfactator" The first Central American BioBrick
Pseudomonas aeruginosa from INDICASAT lab. and the genomic sequence of RhlAB was reviwed in genBank (ID 878955 and 878954) http://www.ncbi.nlm.nih.gov/nuccore/L28170.1
Pseudomonas aeruginosa species of bacteria produce the natural rhamnosyltransferase gene complex (RhlAB). This is the key enzyme responsible for transferring the rhamnose moiety to the b-hydroxyalkanoic acid moiety to biosynthesize rhamnolipid, which is a biomolecule with surfactant properties, but the natural RhlAB gene has illegal restriction sites that make it incompatible with the Assembly Standard Protocol 10, specifically the Pstl1 restriction sites. The design and construction of the rhamnosyltransferase gene into a BioBrick part was the main goal and achievement of the iGEM Panama Team 2010, but since the beginning of this year???s iGEM project we failed in producing the rhamnolipid compound into our E. coli based-factory, because our last year???s BioBrick appeared to have been denaturalized. We then ordered our BioBrick part BBa_K424018 (iGEM Panama team 2010) from The Registry and once it arrived we ran some tests and we noticed that the part was no longer fitted into the plasmid backbone. As scientist and iGEMers, we have the responsibility to deliver a functional BioBrick, thus according to iGEM???s competition new slogan, ???Quality not Quantity???, we have embraced the challenge of re-designing and re-building the same BioBrick part from last year in order to comply this new standard based on quality. How to use it as a BioBrick? : We developed this standarized part to be inserted into E. coli for rhamnolipid production through the mutation of the gene that is responsible to synthesize the key enzyme rhamnosyltranferase (RhlAB) to be compatible with Assembly Standard 10. We have achieved this by performing several silent mutations using the QuikLighting Multi Site-Directed Mutagenesis Kit from Stratagene. This rhamnosyltransferase BioBrick (Rh1AB_BB) is ready to be tested on an expression plasmid device following the Assembly Standard Protocol 10.
The RhlAB gene complex has shown illegal restriction sites according to the assembly standard protocol 10. We faced this problem by applying the mutagenesis protocol that consist in doing silent mutations that change the nucleotides of our rhlAB gene that are cut by the Pstl restriction enzyme. This will give us a standardized coding sequence of the rhlAB gene compatible with the Assembly Standard protocol 10 and a new BioBrick part for the Registry.
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