Team:Edinburgh/Project/Results/Metal promoters Results

Fur Box Assembly

Using the protocol found in the appendix, a 3-part construct was assembled with an acceptor vector pSB1C3 (BBa_K1122009), a Lac promoter and truncated gene called PLac_LacZ (BBa_J33207) optimised for GenBrick and a fluorescent protein, GFP (BBa_K1122004). More information on the structure of the assembly can be found in the GenBrick in action section.

When the assembly was finished, two transformations were made with competent E. coli JM109 and plated on IPTG + chloramphenicol plates and left overnight. In one plate, 4 out of 9 colonies were green fluorescent; the other five had religated and were red fluorescent. Figure 1 shows this plate after two GFP+ colonies were taken and restreaked; the red colonies exhibit RFP from the religated vector. The other plate had a success rate of 8 out of 20 (the competent cells were concentrated x10 before plating). Figure 2 shows the assembly fluorescing under UV when no iron was present, which proves that GFP is successfully over-expressed. Figure 3 shows the gel of restriction-digestion gel using PstI and XbaI.

Figure 1. This plate has chloramphenicol and IPTG. Originally it had 4 out of 9 colonies green fluorescent and the rest red fluorescent, however two green fluorescent colonies were taken and restreaked for the iron assay.