III. Conclusion
[lalaala]
IV. Future Plans
Phase II - Wild type (RR1), RFP-labeled kanR, and GFP-labeled T4MO/Bcr mix
Our ultimate goal is to boost the selection efficiency by introducing a T4MO/Bcr strain, which can interfere with the indole charity work. The Bcr gene, which encodes a multidrug pump, keep this strain survive and produce T4MO. By adjusting IPTG concentration, this strain will keep working for a certain period of time and die afterwards as to the accumulation of kanamycin inside the cell. However, as we didn’t have time to characterize the efficiency of Bcr, and another essential part of our project, the alternative selection method, is still in progress yet, we are unable to do this construction and perform further testing.
(insert a picture here showing out ideal construction)
By having this strain in the population, the charity work will be restricted, so that the selection process can be done efficiently without applying over dosage of antibiotics, and the presence of this strain can be controlled by us so that this alien strain only performs the duty of degrading indole and brings no side effect on the whole selection process.
V. Biobrick construction
Bcr
Bcr is a type of multidrug efflux pump, which are integral membrane proteins that utilize cellular energy to extrude antibiotics or biocides actively out of the cell. It belongs to the major facilitator superfamily (MFS), and is known to contribute to multidrug resistance in E. coli.
Under normal growth conditions, a large number of drug efflux pumps are thought to be weakly expressed. In particular, literature documents Bcr to confer varying degrees of resistance to several kinds of antibiotics when overexpressed; including bicyclomycin (selection-capable), tetracycline (8-fold MIC increase*), and kanamycin (4-fold MIC increase*).
In our iGEM project, we planned to construct a biobrick with the pLac promoter driving expression of Bcr. The reason behind this is to take advantage of the additive effect of IPTG on pLac activation. We hope that by varying the concentration of IPTG, we can control the level of expression of Bcr and thus manipulate the mutant E. coli’s MIC to certain antibiotics.
However, as the time limited,we only submit a plasmid contain only the bcr gene to part registry. You can use it for selection of bacteria in the future.
*: compared with wild type
VI. Appendix
[Extra data] Protocols will probably be included in the Notebook section
[1] (http://www.nature.com/nature/journal/v467/n7311/abs/nature09354.html)
[2](http://www.scielo.br/pdf/gmb/v26n2/a17v26n2.pdf)
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