Part:BBa_K4591009:Design
T500-RFP-lox66-Hpall-lox71-XylSmut-tetO-sfGFP-T500
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2580
Illegal PstI site found at 2574 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2580
Illegal PstI site found at 2574 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2580
Illegal XhoI site found at 2261 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2580
Illegal PstI site found at 2574 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2580
Illegal PstI site found at 2574
Illegal NgoMIV site found at 2255
Illegal AgeI site found at 73
Illegal AgeI site found at 185 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
Our system seems to be able to work perfectly in the environment, but we have to consider that the trigger conditions of the suicide switch are not suitable for the stage of cultivation and modification of engineered bacteria, since the concentrations of engineered bacteria need to be high at that stage. Therefore, it is imperative to investigate novel methods for regulating the suicide switch. The method of homologous recombination also brought us into contacting with the topic of recombinases. Cre-Loxp recombination is a site-specific recombinase technology, which was used to carry out deletions, insertions, translocations and inversions at specific sites in the DNA of cells. The effectiveness of this technology hinges on the presence of flanking sequences, known as Recombinase Target Sites (RTSs), that mark recombination sites. If the RTSs are arranged in a head-to-tail fashion, the area within the RTSs is excised (called resolution) and both RTSs are fused. If the RTSs are arranged in head-to-head orientation, the sequence within the RTSs is flipped (called inversion). With this Flip Module, we can precisely control the on and off of the Suicide Switch by ingenious genetic circuit design and inducing the expression of cre recombinase.
Source
Synthesized by the Tsingke Biotechnology Co., Ltd.
References
[1] Li, J., Nina, M. R. H., Zhang, X., & Bai, Y. (2022). Engineering Transcription Factor XYLS for sensing phthalic acid and terephthalic acid: an application for enzyme evolution. ACS Synthetic Biology, 11(3), 1106–1113. https://doi.org/10.1021/acssynbio.1c00275 [2] Westers, L., Dijkstra, D.S., Westers, H., van Diji, J.M., Quax, W.J. (2006). "Secretion of functional human interleukin-3 from Bacillus subtilis." Journal of biotechnology 123.2 (2006): 211-224. [3] Liu S. L., Du K. (2012). Enhanced expression of an endoglucanase in Bacillus subtilis by using the sucrose-inducible sacB promoter and improved properties of the recombinant enzyme. Protein Expr Purif. 2012 Jun;83(2):164-8. doi: 10.1016/j.pep.2012.03.015. Epub 2012 Apr 4 [4] Guan C., Cui W., Cheng J., Zhou L., Guo J., Hu X., Xiao G., Zhou Z. (2015). Construction and development of an auto-regulatory gene expression system in Bacillus subtilis. Microb Cell Fact. 2015; 14: 150. doi: 10.1186/s12934-015-0341-2. [5] Chen, J., Gai, Y., Fu, G., Zhou, W., Zhang, D., & Wen, J. (2015). Enhanced extracellular production of α-amylase in Bacillus subtilis by optimization of regulatory elements and over-expression of PrsA lipoprotein. Biotechnology letters, 37(4), 899-906. [6] Ansari, A., Ahmed, A. K., Matsangos, A. E., Lay, F., Born, L. J., Marti, G., Harmon, J. W., & Sun, Z. (2016). Cellular GFP toxicity and Immunogenicity: Potential confounders in in vivo cell tracking experiments. Stem Cell Reviews and Reports, 12(5), 553–559. https://doi.org/10.1007/s12015-016-9670-8