Difference between revisions of "Part:BBa K346021"

Latest revision as of 20:12, 27 October 2010

PmerT promoter mutant 3+RBS(B0030)+GFP(E0040)Terminator(B0010)+Terminator(B0012)

We constructed this part to calculate the expression level of GFP downstream of the pmerT mutant 3. The response curve was characterized by using different concentration of Hg(II) to induce GFP's expression and measured the expression level. In detail, when the concentration of Hg(II) in the cytosol changes, the active form of merR dimer will increase and the possibility that it will active pmerT promoter will increase. The strain we used contained the J23103-merR(BBa_K346009) in backbone pSB1A2 and this part was in backbone pSB3K3

Figure 1: The GFP intensity when cultured by different concentration of Hg(II). Cultures were diluted 1:100 in LB and grown to final OD600 0.6. Then different concentration of Hg(II) was added to the culture and we put the tube in the shaker for 2 hours in 30 ℃. Then we suspended the culture with 1% PBS and measure the GFP intensity using a microplate reader.

Construction of merOP Library

Considering the structure of PmerT, it could be expected that mutation at the dyad domain II would change the Ka of MerR-DNA interactions. Thus we mutated the semiconserved sites and left the conserved sites constant.

The approach is generally based upon degenerate primer PCR, with the combination of a ‘DNA shuffling’ procedure, that is performed on the target DNA sequence; the resulting library of variants is then screened for the desired feature, and selected isolates are subjected to a repeated procedure(Fig.6).

Figure.1. Library construction. We performed a degenerate primer PCR on the merTPCAD wild-type promoter region of the alterable sites. The resulting PCR fragments, each potentially contained one or more mutation sites at a restricted location.

Figure.2. Dose-response curves of mutants. Among all these candidates, mutant1, 3, 25, 44, 85, 88 (also shown in Fig 3) were selected for the final careful characterization during which a higher concentration resolution was exploited. Then the dose-response curves were fitted by Hill function.

Figure.3 Sequence comparison of the mutants

It can be observed that mutations at the semiconserved region of PmerT promoter significantly influenced the response behavior of MerR/PmerT pair(Fig.7). It is probably because that the mutations alter the binding affinity between MerR and PmerT promoter. As a result of this, different sensitivity (higher or lower than the wild-type) PmerT promoters were gained by us.

Sequence and Features