Reporter

Part:BBa_K1151038

Designed by: Davide Magr   Group: iGEM13_UniSalento_Lecce   (2013-09-28)
Revision as of 12:52, 2 October 2013 by LeleBiotec (Talk | contribs) (Fluorescence decay assay)

Double generator NikR-GFP, IPTG-nickel regulated

A simple construct composed of BBa_K1151006 + BBa_K1151011, used for the NikR and its responsive promoters activity study.

Fluorescence decay assay

The experiment


                                Beute1.jpg

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

1038.2.jpg 1038.1.jpg


Gfp3.jpg Gfp4.jpg


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

Boiling prep and digestion with EcoRI

Once inserted the construct in a PSB1C3 plasmid (digestion and ligation), we transformed DH5a cells and then we recovered the ligated plasmid by boiling prep. We then proceeded with the digestion with EcoRI.

                              ECO.jpg

Figure 2: Digestion result of boiling prep plasmids.

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


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Categories
Parameters
n/aDouble generator NikR-GFP, IPTG-nickel regulated