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

The greatest obstacle to creating a working kill switch was the fact that it proved impossible to create a part actively expressing MazF; the ligation-transformation process for this part failed consistently. The possibility was considered that the MazF produced under a constitutive promoter was simply too much for even non-deficient cells to handle properly. We therefore created a cloning strain that overproduces MazE. In order to do so, we made use of the ASKA cloning library^{x, a collection of E. coli strains with inducibly overexpressed genes.}

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.↩