Part:BBa_K346096
PmerT promoter-GFP(E0840)
This part was designed to characterize PmerT. When this plasmid was transformed to the bacteria expressing its correspondent activator MerR, the strain would be induced by Hg(II) to express GFP. The relationship between GFP intensity and the Hg(II)'s concentration helped us learn the character of PmerT. The strain we used contained the J23103-merR(BBa_K346009) in backbone pSB1A2 and this part was in backbone pSB3K3.
Figure.1. Hg(II) induced response curve of PmerT-GFP 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.
We also characterized the time and dose response curve of GFP and OD600 of the strain.
Figure.2. Time and dose response of GFP and OD 600. The data was measured every 20 minutes since 20 minutes before supplement of different dose of Hg(II) to the wells and the plates was incubated in the shaker at 37℃during the interval of measurement. For both plates, the volume of LB medium with bacteria was 100uL per well. A: GFP intensity was measured by Tecan Microplate Reader with excitation wavelength at 470nm and emission wavelength at 509nm. A black 96-well plate was used to minimize the interference of different well. B: OD 600 was also measured by Tecan Microplate Reader in a transparent 96-well plate, since the depth of 100uL in the well did not reach 1 centimeter, the OD 600 value here was smaller than that was measured by a spectrophotometer.
Part BBa_K34020-BBa_K34025 are 6 mutants of this part. We constructed a library of PmerT promoter with 6 mutants carefully characterized.
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.3. 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.4. 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.5. 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 730
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