Part:BBa_K1151038

Double generator NikR-GFP, IPTG-nickel regulated

A simple construct composed of K1151006 + K1151011, used for the NikR activity and its responsive promoters study.

Fluorescence decay assay

The experiment

We have set up this experiment to evaluate the shutdown of the fluorescence signal (using fluorimetry technique), adding a fixed quantity of IPTG and nickel to BL21 cells transformed with the plasmid containing this part. Then, we exploited the ability of Olo-NikR to bind the double divergent promoter nikR-exbB (pnikR, pexbB) and repress the transcription of GFP (Nickel added 0.3 ul of stock 10 ug / ul; IPTG: 5 ul 1M).

Discussion

In both graphs it's evident the difference between the 3 controls sample (LB, LB+Ni2+, LB+IPTG) compared to the Ni-IPTG incubated sample in the time-dependent fluorescence signal variations.

The expression of the repressor, NikR (BBa_K1151000), under the plac promoter control, has effects on the fluorescence level of GFP only in the culture incubated with nickel. The response of repression of GFP is found in both the constructs with NikR-responsive promoters and it is quite clear despite the long half-life of GFP.

The proof showed the correct operation of the biobricks, in particular the good functionality of the repression mechanism, nickel-dependent, operated by NikR on the two promoters, that composed a divergent intergenic region of Helicobacter pylori, pnikr e pexbB.

Measurements were made, with Tecan Infinite 200 PRO multimode reader, at two typical wavelenghts of GFP to have a complete and secure framework of the experiments.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1737