Difference between revisions of "Part:BBa K1151036"
 
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                            [[File:beute1.jpg]]
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[[File:beute1Unisalento.jpg]]    [[File:beutafluo2Unisalento.jpg]]
  
 
'''Figure 1:''' Flasks used during the experiment.
 
'''Figure 1:''' Flasks used during the experiment.
  
  
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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.
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We 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 to repress the transcription of GFP.
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(Nickel and IPTG added per flask: 0.3 ul from stock 10 ug / ul;5 ul 1M)
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(Nickel and IPTG added per flask: 0.3 ul from stock 10 ug / ul; 5 ul 1M)
  
  
 
''Results''
 
''Results''
  
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[[File:1036.2.jpg]]                         [[File:1036.3.jpg]]
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[[File:1036.2Unisalento.jpg]]                                   [[File:1036.3Unisalento.jpg]]
  
  
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[[File:decayas.jpg]]                        [[File:gfp2.jpg]]
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[[File:decayasUnisalento.jpg]]                        [[File:gfp2Unisalento.jpg]]
  
 
''Discussion''
 
''Discussion''
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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.
 
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.
  
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===PCR with VF2 and VR2 primers===
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===Other===
  
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                                              [[File:PCR881.jpg]]
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'''PCR with VF2 and VR2 primers'''
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                            [[File:PCR881Unisalento.jpg]]
  
 
'''Figure 2:''' PCR results.
 
'''Figure 2:''' PCR results.

Latest revision as of 17:31, 2 October 2013

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


Beute1Unisalento.jpg Beutafluo2Unisalento.jpg

Figure 1: Flasks used during the experiment.


We 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 to repress the transcription of GFP. (Nickel and IPTG added per flask: 0.3 ul from stock 10 ug / ul; 5 ul 1M)


Results

1036.2Unisalento.jpg 1036.3Unisalento.jpg


DecayasUnisalento.jpg Gfp2Unisalento.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

PCR with VF2 and VR2 primers 

                           PCR881Unisalento.jpg

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