Difference between revisions of "Part:BBa K1151038"
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''Discussion'' | ''Discussion'' | ||
− | In both graphs it's evident the difference between the 3 | + | In both graphs it's evident the difference between the 3 control samples (LB, LB + Ni2+, LB + IPTG) compared to the Ni-IPTG incubated sample regarding variations in the 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 culture 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. |
===Sequence and Features=== | ===Sequence and Features=== |
Revision as of 15:42, 1 October 2013
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
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) compared to the Ni-IPTG incubated sample regarding variations in the 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 culture 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Unknown
- 21INCOMPATIBLE WITH RFC[21]Unknown
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1737