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Team:UniSalento Lecce/Data
From 2013.igem.org
| Line 242: | Line 242: | ||
</section> | </section> | ||
<section id="intro"> | <section id="intro"> | ||
| - | <h3> | + | <h3>2 - HpNikR characterization data</h3> |
<p> | <p> | ||
| - | + | We received this part by Dr. Alberto Danielli, a researcher in molecular biology at the Department of Pharmacology and Biotechnology at the University of Bologna. He sent us the gene encoding HpNikR (from the genome of <i>H. pylori</i> G27) inserted into a pET-15b plasmid. | |
| + | For studying HpNikR, submitted as <a href="http://parts.igem.org/Part:BBa_K1151000">BBa_K1151000</a>, we proceeded with this workflow:</p><br> | ||
| + | |||
| + | <h4><b>HpNikR PCR</b></h4><br> | ||
| + | <img src="https://static.igem.org/mediawiki/parts/4/44/NikRPCR.jpg" height="250px" alt="" align="center" style="margin-left:250px"/> | ||
| + | <br><p>This part was amplified by PCR from the plasmid received by us. | ||
| + | Primers used (including Biobrick Prefix and Suffix, lowercase): | ||
| + | nikRFor: gtttcttcgaattcgcggccgcttctagATGGATACACCCAATAAAGACG | ||
| + | nikRRev: gtttcttcctgcagcggccgctactagtattattaCTATTCATTGTGTTCAAAG</p><br> | ||
| + | |||
| + | <p><b>NikR expression using BL21 (DE3) cells</b></p><br> | ||
| + | Foto: rit | ||
| + | First we made competent BL21 cells and we transformed it with the plasmid containing NikR. We then proceeded with the normal protocol of induction with IPTG for a time of 1, 2 and 4 hours. | ||
| + | |||
| + | <p><b>Cytosol/membrane separation by Zerial method</b></p><br> | ||
| + | Foto: 42 | ||
| + | To confirm that NikR is a cytosolic protein (not expressed in multivesicular bodies, and then in membrane) we performed a separation membrane-cytosol (Zerial method) (sample: 2-hours induced cells). | ||
| + | |||
| + | <p><b>NikR purification by Ni-NTA resin</b></p><br> | ||
| + | Foto: 41 | ||
| + | NikR (sample: 2-hours induced cells) can be purified by Ni-NTA resin, which has a high affinity for histidine residues. | ||
| + | |||
| + | <p><b>NikR incubation with nickel sulfate and Sephadex molecular exclusion chromatography</b></p><br> | ||
| + | Foto: resina | ||
| + | We therefore decided to study the NikR protein: first, we focused on its characteristic to bind nickel. We have developed a protocol of incubation of the protein with nickel sulfate (stock: 10 ug/ul), in presence of an Incubation buffer (20 mM Tris pH 7,6, 100 mM NaCl). These are the samples we tested (each has a final volume of 100 ul): | ||
| + | 1. 1,2 ul (1 ug) NikR + 0,3 ul Nickel sulfate + 98,5 ul Incubation buffer | ||
| + | 2. 6,1 ul (5 ug) NikR + 1,5 ul Nickel sulfate + 92,4 ul Incubation buffer | ||
| + | 3. 12,2 ul (10 ug) NikR + 3 ul Nickel sulfate + 84,8 ul Incubation buffer | ||
| + | The samples were put on wheel at 4°C overnight. | ||
| + | In order to eliminate the nickel eccess we proceeded with a molecular exclusion chromatography on Sephadex G-25 resin. | ||
| + | In this way the protein with the nickel-binding sites saturated will be released first from the column, collecting the eluate easily. The nickel in excess will remain trapped in the pores of the resin. | ||
| + | |||
| + | <p><b>ICP-AES assay</b></p><br> | ||
| + | Foto: machinery | ||
| + | Inductively-Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) is a type of emission spectroscopy that uses the inductively coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a particular element. The intensity of this emission is indicative of the concentration of the element within the sample. These are the samples (also here the final volume of each is 100 ul) that we have analyzed: | ||
| + | 1. 100 ul Incubation buffer | ||
| + | 2. 1,5 ul Nickel sulfate + 98,5 ul Incubation buffer | ||
| + | 3. 1,2 ul (1 ug) NikR + 0,3 ul Nickel sulfate + 98,5 ul Incubation buffer | ||
| + | 4. 6,1 ul (5 ug) NikR + 1,5 ul Nickel sulfate + 92,4 ul Incubation buffer | ||
| + | 5. 12,2 ul (10 ug) NikR + 3 ul Nickel sulfate + 84,8 ul Incubation buffer | ||
| + | 6. Sephadex resin after incubation with the sample n.4 | ||
| + | Results: | ||
| + | Foto: tabella1, icp2, icp3 | ||
| + | Discussion: | ||
| + | Although it remains difficult to identify accurately the binding NikR-nickel stoichiometry: we can say, however, that between the two variables exists a direct relationship. | ||
| + | |||
| + | <p><b>ATR-FTIR assay</b></p><br> | ||
| + | Foto: ATR | ||
| + | To complete our analysis on the protein, we studied the conformational changes which HpNikR goes against in the bond with Ni2+ ions through Attentuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR). With this technique it is possible to acquire the infrared absorption spectra of our control (Apo-HpNikR) and of our protein treated with NiSO4 (complex HpNikR:Ni), so as to obtain the IR differential spectrum. By means of these it will be possible to highlight how some spectrum peaks suffer changes in infrared absorption due to changes in protein conformation induced by the nickel binding. | ||
| + | Our hypothesis: | ||
| + | foto: y3 | ||
| + | The analysis of the spectrum of the native protein shows two peaks at values of wave number of 1261 cm-1 and 800 cm-1; these peaks are absent in the protein in the presence of nickel and everything is confirmed by the differential spectrum.These peaks could be assigned to a tyrosine residue that due to the interaction with nickel undergoes a deprotonation of the hydroxyl functional group. So it can be assumed that the bond to the metal induces a change necessary to the regulator NikR for the interaction with DNA. | ||
</p> | </p> | ||
</section> | </section> | ||
Revision as of 12:57, 2 October 2013
Experimental data and results
1 - Submitted parts agarose gel analysis
Here you can find a PCR and/or a restriction analysis of these parts: BBa_K1151001, BBa_K1151005, BBa_K1151009, BBa_K1151010, BBa_K1151011, BBa_K1151036, BBa_K1151038, made to evaluate their identity. The molecular weight of these parts has been evaluated with the Invitrogen 1kb DNA Ladder (15615-016)
foto
The K1151001 digestion with EcoRI and PstI displays a profile confirming its molecular weight.
foto
foto
The K1151001 PCR with VF2 (BBa_BBa_G00100) and VR (BBa_BBa_G00101) displays a profile confirming its molecular weight.
foto
foto
The K1151005 digestion with EcoRI and PstI displays a profile confirming its molecular weight.
foto
foto
The K1151005 PCR with VF2 (BBa_BBa_G00100) and VR (BBa_BBa_G00101) displays a profile confirming its molecular weight.
foto
foto
The K1151009 digestion with EcoRI and PstI displays a profile confirming its molecular weight.
foto
foto
The K1151009 PCR with VF2 (BBa_BBa_G00100) and VR (BBa_BBa_G00101) displays a profile confirming its molecular weight.
foto
foto
The K1151010 digestion with EcoRI and PstI displays a profile confirming its molecular weight.
foto
foto
The K1151010 PCR with VF2 (BBa_BBa_G00100) and VR (BBa_BBa_G00101) displays a profile confirming its molecular weight.
foto
foto
The K1151011 digestion with EcoRI and PstI displays a profile confirming its molecular weight.
foto
foto
The K1151011 PCR with VF2 (BBa_BBa_G00100) and VR (BBa_BBa_G00101) displays a profile confirming its molecular weight.
foto
foto
The K1151036 PCR with VF2 (BBa_BBa_G00100) and VR (BBa_BBa_G00101) displays a profile confirming its molecular weight.
foto
foto
The K1151038 digestion with EcoRI displays a profile confirming its molecular weight.
foto
2 - HpNikR characterization data
We received this part by Dr. Alberto Danielli, a researcher in molecular biology at the Department of Pharmacology and Biotechnology at the University of Bologna. He sent us the gene encoding HpNikR (from the genome of H. pylori G27) inserted into a pET-15b plasmid. For studying HpNikR, submitted as BBa_K1151000, we proceeded with this workflow:
HpNikR PCR
This part was amplified by PCR from the plasmid received by us. Primers used (including Biobrick Prefix and Suffix, lowercase): nikRFor: gtttcttcgaattcgcggccgcttctagATGGATACACCCAATAAAGACG nikRRev: gtttcttcctgcagcggccgctactagtattattaCTATTCATTGTGTTCAAAG
NikR expression using BL21 (DE3) cells
Foto: rit First we made competent BL21 cells and we transformed it with the plasmid containing NikR. We then proceeded with the normal protocol of induction with IPTG for a time of 1, 2 and 4 hours.
Cytosol/membrane separation by Zerial method
Foto: 42 To confirm that NikR is a cytosolic protein (not expressed in multivesicular bodies, and then in membrane) we performed a separation membrane-cytosol (Zerial method) (sample: 2-hours induced cells).
NikR purification by Ni-NTA resin
Foto: 41 NikR (sample: 2-hours induced cells) can be purified by Ni-NTA resin, which has a high affinity for histidine residues.
NikR incubation with nickel sulfate and Sephadex molecular exclusion chromatography
Foto: resina We therefore decided to study the NikR protein: first, we focused on its characteristic to bind nickel. We have developed a protocol of incubation of the protein with nickel sulfate (stock: 10 ug/ul), in presence of an Incubation buffer (20 mM Tris pH 7,6, 100 mM NaCl). These are the samples we tested (each has a final volume of 100 ul): 1. 1,2 ul (1 ug) NikR + 0,3 ul Nickel sulfate + 98,5 ul Incubation buffer 2. 6,1 ul (5 ug) NikR + 1,5 ul Nickel sulfate + 92,4 ul Incubation buffer 3. 12,2 ul (10 ug) NikR + 3 ul Nickel sulfate + 84,8 ul Incubation buffer The samples were put on wheel at 4°C overnight. In order to eliminate the nickel eccess we proceeded with a molecular exclusion chromatography on Sephadex G-25 resin. In this way the protein with the nickel-binding sites saturated will be released first from the column, collecting the eluate easily. The nickel in excess will remain trapped in the pores of the resin.
ICP-AES assay
Foto: machinery Inductively-Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) is a type of emission spectroscopy that uses the inductively coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a particular element. The intensity of this emission is indicative of the concentration of the element within the sample. These are the samples (also here the final volume of each is 100 ul) that we have analyzed: 1. 100 ul Incubation buffer 2. 1,5 ul Nickel sulfate + 98,5 ul Incubation buffer 3. 1,2 ul (1 ug) NikR + 0,3 ul Nickel sulfate + 98,5 ul Incubation buffer 4. 6,1 ul (5 ug) NikR + 1,5 ul Nickel sulfate + 92,4 ul Incubation buffer 5. 12,2 ul (10 ug) NikR + 3 ul Nickel sulfate + 84,8 ul Incubation buffer 6. Sephadex resin after incubation with the sample n.4 Results: Foto: tabella1, icp2, icp3 Discussion: Although it remains difficult to identify accurately the binding NikR-nickel stoichiometry: we can say, however, that between the two variables exists a direct relationship.
ATR-FTIR assay
Foto: ATR To complete our analysis on the protein, we studied the conformational changes which HpNikR goes against in the bond with Ni2+ ions through Attentuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR). With this technique it is possible to acquire the infrared absorption spectra of our control (Apo-HpNikR) and of our protein treated with NiSO4 (complex HpNikR:Ni), so as to obtain the IR differential spectrum. By means of these it will be possible to highlight how some spectrum peaks suffer changes in infrared absorption due to changes in protein conformation induced by the nickel binding. Our hypothesis: foto: y3 The analysis of the spectrum of the native protein shows two peaks at values of wave number of 1261 cm-1 and 800 cm-1; these peaks are absent in the protein in the presence of nickel and everything is confirmed by the differential spectrum.These peaks could be assigned to a tyrosine residue that due to the interaction with nickel undergoes a deprotonation of the hydroxyl functional group. So it can be assumed that the bond to the metal induces a change necessary to the regulator NikR for the interaction with DNA.
title
Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id quod mazim placerat facer possim assum. Typi non habent claritatem insitam; est usus legentis in iis qui facit eorum claritatem. Investigationes demonstraverunt lectores legere me lius quod ii legunt saepius. Claritas est etiam processus dynamicus, qui sequitur mutationem consuetudium lectorum. Mirum est notare quam littera gothica, quam nunc putamus parum claram, anteposuerit litterarum formas humanitatis per seacula quarta decima et quinta decima. Eodem modo typi, qui nunc nobis videntur parum clari, fiant sollemnes in futurum.
title
Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id quod mazim placerat facer possim assum. Typi non habent claritatem insitam; est usus legentis in iis qui facit eorum claritatem. Investigationes demonstraverunt lectores legere me lius quod ii legunt saepius. Claritas est etiam processus dynamicus, qui sequitur mutationem consuetudium lectorum. Mirum est notare quam littera gothica, quam nunc putamus parum claram, anteposuerit litterarum formas humanitatis per seacula quarta decima et quinta decima. Eodem modo typi, qui nunc nobis videntur parum clari, fiant sollemnes in futurum.
title
Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id quod mazim placerat facer possim assum. Typi non habent claritatem insitam; est usus legentis in iis qui facit eorum claritatem. Investigationes demonstraverunt lectores legere me lius quod ii legunt saepius. Claritas est etiam processus dynamicus, qui sequitur mutationem consuetudium lectorum. Mirum est notare quam littera gothica, quam nunc putamus parum claram, anteposuerit litterarum formas humanitatis per seacula quarta decima et quinta decima. Eodem modo typi, qui nunc nobis videntur parum clari, fiant sollemnes in futurum.
title
Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id quod mazim placerat facer possim assum. Typi non habent claritatem insitam; est usus legentis in iis qui facit eorum claritatem. Investigationes demonstraverunt lectores legere me lius quod ii legunt saepius. Claritas est etiam processus dynamicus, qui sequitur mutationem consuetudium lectorum. Mirum est notare quam littera gothica, quam nunc putamus parum claram, anteposuerit litterarum formas humanitatis per seacula quarta decima et quinta decima. Eodem modo typi, qui nunc nobis videntur parum clari, fiant sollemnes in futurum.
title
Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id quod mazim placerat facer possim assum. Typi non habent claritatem insitam; est usus legentis in iis qui facit eorum claritatem. Investigationes demonstraverunt lectores legere me lius quod ii legunt saepius. Claritas est etiam processus dynamicus, qui sequitur mutationem consuetudium lectorum. Mirum est notare quam littera gothica, quam nunc putamus parum claram, anteposuerit litterarum formas humanitatis per seacula quarta decima et quinta decima. Eodem modo typi, qui nunc nobis videntur parum clari, fiant sollemnes in futurum.
title
Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id quod mazim placerat facer possim assum. Typi non habent claritatem insitam; est usus legentis in iis qui facit eorum claritatem. Investigationes demonstraverunt lectores legere me lius quod ii legunt saepius. Claritas est etiam processus dynamicus, qui sequitur mutationem consuetudium lectorum. Mirum est notare quam littera gothica, quam nunc putamus parum claram, anteposuerit litterarum formas humanitatis per seacula quarta decima et quinta decima. Eodem modo typi, qui nunc nobis videntur parum clari, fiant sollemnes in futurum.
title
Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id quod mazim placerat facer possim assum. Typi non habent claritatem insitam; est usus legentis in iis qui facit eorum claritatem. Investigationes demonstraverunt lectores legere me lius quod ii legunt saepius. Claritas est etiam processus dynamicus, qui sequitur mutationem consuetudium lectorum. Mirum est notare quam littera gothica, quam nunc putamus parum claram, anteposuerit litterarum formas humanitatis per seacula quarta decima et quinta decima. Eodem modo typi, qui nunc nobis videntur parum clari, fiant sollemnes in futurum.
title
Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id quod mazim placerat facer possim assum. Typi non habent claritatem insitam; est usus legentis in iis qui facit eorum claritatem. Investigationes demonstraverunt lectores legere me lius quod ii legunt saepius. Claritas est etiam processus dynamicus, qui sequitur mutationem consuetudium lectorum. Mirum est notare quam littera gothica, quam nunc putamus parum claram, anteposuerit litterarum formas humanitatis per seacula quarta decima et quinta decima. Eodem modo typi, qui nunc nobis videntur parum clari, fiant sollemnes in futurum.
title
Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id quod mazim placerat facer possim assum. Typi non habent claritatem insitam; est usus legentis in iis qui facit eorum claritatem. Investigationes demonstraverunt lectores legere me lius quod ii legunt saepius. Claritas est etiam processus dynamicus, qui sequitur mutationem consuetudium lectorum. Mirum est notare quam littera gothica, quam nunc putamus parum claram, anteposuerit litterarum formas humanitatis per seacula quarta decima et quinta decima. Eodem modo typi, qui nunc nobis videntur parum clari, fiant sollemnes in futurum.
