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 9: / Line 9: @@
-                                      [[File:beute1.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 (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 double divergent promoter nikR-exbB (pnikR, pexbB) and repress the transcription of GFP (Nickel added 0.3 ul of stock 10 ug / ul; IPTG: 5 ul 1M). All this was made possible through a series of measurements conducted at fluorometer.
+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:1038.2.jpg]]                           [[File:1038.1.jpg]]
+[[File:1038.2Unisalento.jpg]]                           [[File:1038.1Unisalento.jpg]]
-[[File:gfp3.jpg]]                             [[File:gfp4.jpg]]
+[[File:gfp3Unisalento.jpg]]                             [[File:gfp4Unisalento.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 <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===
-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.
+'''Boiling prep and digestion with EcoRI'''
-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.
+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]]
-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.
+'''Figure 2:''' Digestion result of boiling prep plasmids.
 ===Sequence and Features===