Part:BBa_K1151036

Double generator NikR-GFP, IPTG-nickel regulated

A simple construct composed of the parts BBa_K1151006 + BBa_K1151009, used for the NikR and its responsive promoters activity study.

Fluorescence decay assay

The experiment

Figure 1: Flasks used during the experiment.

We have set up this experiment to evaluate the shutdown of the fluorescence signal, through a series of measurements conducted at fluorometer. We added a fixed quantity of IPTG and nickel to BL21 cells transformed with the plasmid containing this part, and exploited the ability of Olo-NikR to bind the pnikR promoter and repress the transcription of GFP. (Nickel and IPTG added per flask: 0.3 ul from stock 10 ug / ul;5 ul 1M)

Results

Discussion

In both graphs it's evident the difference between the 3 control samples (LB, LB + Ni2+, LB + IPTG) and the Ni-IPTG incubated sample regarding variations in time-dependent fluorescence signal. The expression of the repressor, NikR (BBa_K1151000), under the control of a pLac promoter, has effects on the fluorescence level of GFP only in the cultures incubated with nickel. The response of repression of GFP is found in both the constructs (BBa_K1151036 and BBa_K1151038) 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 and pexbB. Measurements were made, with a Tecan Infinite 200 PRO multimode reader, at two typical wavelenghts of GFP in order to have a complete and secure framework of the experiments.

Other

PCR with VF2 and VR2 primers

Figure 2: PCR results.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 818
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 818
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 818
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 818
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 818
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1616