Part:BBa_K4060000:Experience

1. Cloning MazE and MazF gene

The MazE and MAzF have been amplified by PCR from E. coli MG1655 genome DNA.

Fig.1 Agarose electrophoresis in order to verify MazE and MazF genes. The purified DNA fragments had the expected length and were subsequently used for cloning. Lane 1, 100bp ladder. Lane 2, MazE PCR product. Lane 3 and 4, MazF PCR product. Land 5, 100bp ladder.

2. Gibson Assembly

Fig. 2 Agarose electrophoresis in order to verify Construct 1 and Construct 2. Lane 1, 100bp ladder. Lane 1' partial of Life 1 kb Plus ladder. Lane 2 and 3, Construct 1 digested with BamHI. Lane 4, Construct 2 digested with KpnI and NdeI.

After finding out that putting pBad in the former is the better choice, we generated five other tandem promoters with Strategy 3 to construct them rapidly. In these 5 tandem promoters, we selected five other constitutive promoters (BBa_J23102, BBa_J23118, BBa_J23106, BBa_J23110, BBa_J23113) in the same family of J23106. (Promoter strength：BBa_J23102＞ BBa_J23118＞ BBa_J23106＞ BBa_J23110＞ BBa_J23116>BBa_J23113) Finally, after constructing these tandem promoters, we checked the strength of the tandem promoters by testing the RFP expression from each tandem promoter.

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