Difference between revisions of "Part:BBa K1151038"

Latest revision as of 10:14, 3 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) 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 to check.

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

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1151038 short</partinfo>
-A simple construct composed of K1151006 + K1151011, used for the NikR activity study.
+A simple construct composed of [[BBa_K1151006]] + [[BBa_K1151011]], used for the NikR and its responsive promoters activity study.
 ===Fluorescence decay assay===
-[[File:1038.2.jpg]]                           [[File:1038.1.jpg]]
+''The experiment''
-[[File:gfp3.jpg]]                             [[File:gfp4.jpg]]
+[[File:beute1Unisalento.jpg]]        [[File:beutafluo2Unisalento.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''
+[[File:1038.2Unisalento.jpg]]                           [[File:1038.1Unisalento.jpg]]
+[[File:gfp3Unisalento.jpg]]                             [[File:gfp4Unisalento.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 <i>Helicobacter pylori</i>, 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 to check.
+                               [[File:ECOUnisalento.jpg]]
+'''Figure 2:''' Digestion result of boiling prep plasmids.
 ===Sequence and Features===