Difference between revisions of "Part:BBa K3634012:Design"

Revision as of 11:07, 6 August 2020

Glucose-Mediated Death Sensor

Design Notes

The sequence for O2 was found 401bp downstream of O1, included in the lacZ gene, and was therefore not included as lacZ, lacY and lacA were later replaced by ccdB. The O3 sequence was found to have a 13bp overlap with the CAP binding site therefore was not mutated as this may have affected the CAP binding affinity. The sequence was truncated however where the CAP binding site and O3 5' sequence did not overlap. This was proposed to reduce binding affinity of the repressor. Three mutations were made to the O1 sequence: g91t, t98g and c101g. The native 6bp RBS downstream of lacO1 was also replaced with the weak RBS BBa_B0031 with spacer regions either side left as previous. The ccdB sequence was taken from BBa_K145151 and found to have an illegal BsaI restriction site at 222bp. The appropriate alteration was made to make the part RFC[1000] compatible: c219g (GTC (Val) to got to go (Val)). As native to E.coli, no codon optimisation step was required.

Source

The lac operon with the associated regulatory upstream components are found natively in E.coli. The initial sequence of the lacO1 was taken from Gilbert and Maxam (1973) and later confirmed by Oehler et al. (1990), where other sequences included in this part and appropriate mutations were gathered. The ccdB toxin is also found natively in E.coli and the sequence specified in BBa_K145151 by KULeuven iGEM (2008). This sequence was confirmed against pAG415 GPD-EGFP and pCMV SPORT6ccdB plasmids and in silico site-directed mutagenesis removed a BsaI site so that the part was RFC[1000] standard (BBa_K3634011).

References

Jacob F., Monod J. 1961. Genetic Regulatory Mechanisms in the Synthesis of Proteins. J. Mol. Biol. 3: p818-356. Gilbert W., Maxam A. 1973. The Nucleotide Sequence of the lac Operator. Proc. Nat. Acad. Sci. USA. 70(12): p3581-3584. Oehler S., Eismann E.R., Krämer H., Müller-Hill B. 1990. The three operators of the lac operon cooperate in repression. EMBO J. 9(v): p973-979. Bernard P., Couturier M. 1992. Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes. J. Mol. Biol. 226: p735–745. KULeuven iGEM 2008 - https://parts.igem.org/Part:BBa_K145151 Vandervelde A., Drobnak I., Hadži S., Sterckx Y.GJ., Welte T., Greve. H.D., Charlier D., Efremov R., Loris R., Lah J. 2017. Molecular mechanism governing ratio-dependent transcription regulation in the ccdAB operon. NAR. 45(6): p2937-2950.