Part:BBa_K424018:Design
Rhamnosiltransferase BioBrick (Rh1AB_BB)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 169
Illegal BamHI site found at 729
Illegal XhoI site found at 905
Illegal XhoI site found at 2191 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1084
Illegal NgoMIV site found at 1805
Illegal NgoMIV site found at 1918 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 394
Illegal BsaI site found at 1434
Illegal BsaI.rc site found at 578
Design Notes
The RhlAB gene complex have shown illegal restriction sites by the restriccition enzymes of the assembly standard protocol 10. We face this problem by applying the mutagenesis protocol that consist in doing silent mutation that change the nucleotides of our rhlAB gene that is cutted by the Pstl restriction enzyme. Then we will get an standardized coding sequence of the rhlAB gene compatible with the Assembly Standard 10.
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
References
Qinhong Wang, Xiangdong Fang, Baojun Bai, Xiaolin Liang, Patrick J. Shuler, William A. Goddard III, Yongchun Tang. 2007. Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery. Biotechnology and Bioengineering Volume 98, Issue 4, pages 842–853.
Ochsner UA, Fiechter A, Reiser J. 1994. Isolation, characterization, and expression in Escherichia coli of the Pseudomonas aeruginosa rhlAB genes encoding a rhamnosyltransferase involved in rhamnolipid biosurfactant synthesis. J Biol Chem 269:19787–19795.
Maier RM, Soberon-Chavez G. 2000. Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications. Appl Microbiol Biotechnol 54(5):625–633.
QuikChange Lightning Multi Site-Directed Mutagenesis Kit Instruction manual, catalog #210515 from Stratagene.