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 the parts[[ Bba_K1151006]] + [[Bba_K1151009]], used for the NikR and its responsive promoters 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''
-We have set up this experiment to evaluate the shutdown of the fluorescence signal (using fluorimetry technique), adding a fixed quantity of IPTG and nickel to BL21 cells transformed with the plasmid containing this part. Then, we exploited the ability of Olo-NikR to bind the promoter pnikR and repress the transcription of GFP (Nickel added 0.3 ul of stock 10 ug / ul; IPTG: 5 ul 1M).
+[[File:beute1Unisalento.jpg]]    [[File: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''
-[[File:1036.2.jpg]]                          [[File:1036.3.jpg]]
+[[File:1036.2Unisalento.jpg]]                                    [[File:1036.3Unisalento.jpg]]
-[[File:decayas.jpg]]                         [[File:gfp2.jpg]]
+[[File:decayasUnisalento.jpg]]                         [[File:gfp2Unisalento.jpg]]
 ''Discussion''
-In both graphs it's evident the difference between the 3 controls sample (LB, LB+Ni2+, LB+IPTG) compared to the Ni-IPTG incubated sample in the time-dependent fluorescence signal variations.
+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.
-The expression of the repressor, NikR (BBa_K1151000), under the plac promoter control, 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 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 e pexbB.
+===Other===
-Measurements were made, with Tecan Infinite 200 PRO multimode reader, at two typical wavelenghts of GFP to have a complete and secure framework of the experiments.
+'''PCR with VF2 and VR2 primers'''
+                            [[File:PCR881Unisalento.jpg]]
+'''Figure 2:''' PCR results.
 ===Sequence and Features===