Team:Wageningen UR/Notebook/OptogeneticKillSwitch
These experiments were performed primarily by Marijn, with significant contributions by Mark during the final phase. Unless otherwise indicated, protocols were done according to the Protocols page.
May 16 - June 6: Obtaining Parts
Both MazE and MazF have been registered as BioBricks (parts BBa_K1096001 and BBa_K302033, respectively). However, as of the creation of the kill switch, neither are available from the iGEM registry. For this reason, we chose to isolate both genes directly from E. coli genomic DNA via PCR. In addition, the following BioBricks were prepared from iGEM registry kits:
- BBa_K592006: FixK2 (Promoter triggered by YF1-FixJ)
- BBa_K592016: YF1-FixJ (Light sensor)
- BBa_B0015: Double terminator
- BBa_J23108: Constituitive promoter of the Anderson family (Mid-level expression strength, arbitrarily chosen.)
Accounting for the possibility that natively produced MazE could interfere with the kill switch, we also obtained a MazEF-deficient strain (MC4100ΔmazEFrelA+, or ΔmazEF for short)1 for potential use as a chassis organism. A culture of these cells was made chemically competent.
4 July - 1 August: pDawn Construction
8 August - 3 October: pDusk Construction
References
1. Sat, B., Reches, M., Engelberg-Kulka, H. (2003)
The Escherichia coli mazEF Suicide Module Mediates Thymineless Death. Journal of Bacteriology vol. 185 no. 6, 1803-1807.
↩
2.Kitagawa, M., Ara, T., Arifuzzaman, M,, Ioka-Nakamichi, T., Inamoto, E., Toyonaga, H., Mori, H. (2005) Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Research 12(5):291-9.↩
