Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Team Panama 2011
Date created: 2011-09-25 11:00:00
Date modified: 2015-05-08 01:13:00
Re-designing "The Biosurfactator" The first Central American BioBrick
| Types | DnaRegion |
| Roles | Coding CDS |
| Sequences | BBa_K653000_sequence (Version 1) |
Description
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.
Notes
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.Source
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
| Sequence Annotation | Location | Component / Role(s) |
| BBa_ K653000 RhlAB Panama Primer Rhamnosyltransferase gene complex 1 Rhamnosyltranferase A Silent Mutation Rhamnosyltransferase B Silent Mutation Silent Mutation Primer | 1,2314 1,19 35,2234 35,922 754,759 988,2268 1777,1782 1891,1896 2296,2314 | engineered_region feature/BioBrick sequence_feature feature/misc feature/dna sequence_feature CDS feature/cds point_mutation feature/s_mutation CDS feature/cds feature/s_mutation point_mutation feature/s_mutation point_mutation sequence_feature feature/misc |