Difference between revisions of "Part:BBa K1151036"

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

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

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

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1151036 short</partinfo>
-A simple construct composed of K1151006 + K1151009, used for the NikR activity study.
+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''
-[[File:1036.2.jpg]]                          [[File:1036.3.jpg]]
+[[File:beute1Unisalento.jpg]]    [[File:beutafluo2Unisalento.jpg]]
+'''Figure 1:''' Flasks used during the experiment.
-[[File:decayas.jpg]]                         [[File:gfp2.jpg]]
+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''
+[[File:1036.2Unisalento.jpg]]                                    [[File:1036.3Unisalento.jpg]]
+[[File:decayasUnisalento.jpg]]                         [[File: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'''
+                            [[File:PCR881Unisalento.jpg]]
+'''Figure 2:''' PCR results.
 ===Sequence and Features===