Team:UniSalento Lecce/Data

From 2013.igem.org

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      <li><a href="#o3" style="text-decoration: none"><i class="icon-chevron-right"></i>3. Storage system (link dallo slider STORE IT)</a>
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      <li><a href="#o3" style="text-decoration: none"><i class="icon-chevron-right"></i>3. Nickel responsive promoters characterization data</a>
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      <li class="caption"><a href="#o3_1" style="text-decoration: none; color: #777777;">1. Hpn: a storage protein in H. pylori</a></li>
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      <li class="caption"><a href="#o3_1" style="text-decoration: none; color: #777777;">1. pnikR</a></li>
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                                                        <li class="caption"><a href="#o3_1" style="text-decoration: none; color: #777777;">2. Double divergent promoter (pnikR-pexbB)</a></li>
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      <li><a href="#o4" style="text-decoration: none"><i class="icon-chevron-right"></i>4. Nickel-dependant Quorum Sensing Network</a></li>
 
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      <li><a href="#o5" style="text-decoration: none"><i class="icon-chevron-right"></i>5. General cloning and working schemes</a></li>
 
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      <li><a href="#o6" style="text-decoration: none"><i class="icon-chevron-right"></i>6. Bibliography</a></li>
 
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<h4><b>2.7 - CryoTEM microscopy of purified HpNikR</b></h4><br>
<h4><b>2.7 - CryoTEM microscopy of purified HpNikR</b></h4><br>
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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.
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High quality of the CryoTEM image displays:
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Correct sample preparation
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Correct set of experimental and strumental conditions
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High probability that the image is relative to purified HpNikR protein. Assuming that ribosomal particles, whose molecular weight is around 200 kDa, have a diameter around 20 nm, the particle of diameter of around 1-2 nm which can be seen may coincide with a particle of MW around 20 kDa. HpNikR MW is around 17 kDa.
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<section id="intro">
<section id="intro">
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<h3>3 - Nickel responsive promoters characterization data</h3><br>
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<h4><b>Fluorescence Decay assay</b></h4><br>
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<p>We set up these experiments 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 and the double divergent promoter, thus repressing the transcription of GFP. (Nickel and IPTG added per flask: 0.3 ul from stock 10 ug / ul; 5 ul 1M)
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<img src="https://static.igem.org/mediawiki/parts/4/43/Beute1Unisalento.jpg" height="250px" alt="" align="center" style="margin-left:25px;margin-bottom:25px;margin-top:0px"/><br>
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                                        <h4><b>3.1 - pnikR</b></h4><br>
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<p>To characterize this part we used <a href="http://parts.igem.org/Part:BBa_K1151036">BBa_K1151036</a>. These data shows the ability of HpNikR to regulate pnikR, regulating GFP expression.<br></p><br>
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<img src="https://static.igem.org/mediawiki/parts/9/91/1036.2Unisalento.jpg" widht="120px" alt="" align="center" style="margin-left:100px;margin-bottom:25px;margin-top:0px"/><br>
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<img src="https://static.igem.org/mediawiki/parts/2/2e/DecayasUnisalento.jpg" height="300px" alt="" align="center" style="margin-left:100px;margin-bottom:25px;margin-top:0px"/><br>
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<img src="https://static.igem.org/mediawiki/parts/7/78/1036.3Unisalento.jpg" widht="120px" alt="" align="center" style="margin-left:100px;margin-bottom:25px;margin-top:0px"/><br>
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<img src="https://static.igem.org/mediawiki/parts/a/a2/Gfp2Unisalento.jpg" height="300px" alt="" align="center" style="margin-left:100px;margin-bottom:25px;margin-top:0px"/><br>
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<h4><b>3.2 - Double divergent promoter pnikR-pexbB</b></h4><br>
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<p>To characterize this part we used <a href="http://parts.igem.org/Part:BBa_K1151038">BBa_K1151038</a>. These data shows the ability of HpNikR to regulate divergent promoter, regulating GFP expression.<br></p><br>
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<img src="https://static.igem.org/mediawiki/parts/4/44/1038.2Unisalento.jpg" widht="120px" alt="" align="center" style="margin-left:100px;margin-bottom:25px;margin-top:0px"/><br>
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<img src="https://static.igem.org/mediawiki/parts/c/cd/Gfp3Unisalento.jpg" height="300px" alt="" align="center" style="margin-left:100px;margin-bottom:25px;margin-top:0px"/><br>
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<img src="https://static.igem.org/mediawiki/parts/1/11/1038.1Unisalento.jpg" widht="120px" alt="" align="center" style="margin-left:100px;margin-bottom:25px;margin-top:0px"/><br>
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<img src="https://static.igem.org/mediawiki/parts/4/49/Gfp4Unisalento.jpg" height="300px" alt="" align="center" style="margin-left:100px;margin-bottom:25px;margin-top:0px"/><br><br>
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</p>
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<p>All the graphs show 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, HpNikR (<a href="http://parts.igem.org/Part:BBa_K1151000">BBa_K1151000</a>), 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 (<a href=”http://parts.igem.org/Part:BBa_K1151036”>BBa_K1151036</a> and <a href=”http://parts.igem.org/Part:BBa_K1151038”>BBa_K1151038</a>) with HpNiKR 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 the two typical wavelenghts of GFP in order to have a complete and secure framework of the experiments.</p>
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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.
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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.
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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.
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Revision as of 17:59, 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)


BBa_K1151001



The K1151001 digestion with EcoRI and PstI displays a profile confirming its molecular weight.






The K1151001 PCR with VF2 (BBa_BBa_G00100) and VR (BBa_BBa_G00101) displays a profile confirming its molecular weight.





BBa_K1151005



The K1151005 digestion with EcoRI and PstI displays a profile confirming its molecular weight.

foto






The K1151005 PCR with VF2 (BBa_BBa_G00100) and VR (BBa_BBa_G00101) displays a profile confirming its molecular weight.







BBa_K1151009




The K1151009 digestion with EcoRI and PstI displays a profile confirming its molecular weight.









The K1151009 PCR with VF2 (BBa_BBa_G00100) and VR (BBa_BBa_G00101) displays a profile confirming its molecular weight.







BBa_K1151010



The K1151010 digestion with EcoRI and PstI displays a profile confirming its molecular weight.










The K1151010 PCR with VF2 (BBa_BBa_G00100) and VR (BBa_BBa_G00101) displays a profile confirming its molecular weight.







BBa_K1151011




The K1151011 digestion with EcoRI and PstI displays a profile confirming its molecular weight.





The K1151011 PCR with VF2 (BBa_BBa_G00100) and VR (BBa_BBa_G00101) displays a profile confirming its molecular weight.





BBa_K1151036




The K1151036 PCR with VF2 (BBa_BBa_G00100) and VR (BBa_BBa_G00101) displays a profile confirming its molecular weight.








BBa_K1151038



The K1151038 digestion with EcoRI displays a profile confirming its molecular weight.









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 the following workflow. Go to the Protocols page


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2.1 - 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








2.2 - HpNikR expression using BL21(DE3) cells






First we made ​​competent BL21 cells and we transformed it with the plasmid containing HpNikR. We then proceeded with the normal protocol of induction with IPTG for a time of 1, 2 and 4 hours.










2.3 - Cytosol/membrane separation by Zerial method





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).












2.4 - HpNikR purification by Ni-NTA resin





NikR (sample: 2-hours induced cells) can be purified by Ni-NTA resin, which has a high affinity for histidine residues.

2.5 - ICP-AES assay


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) analyzed, see the Protocols page for Incubation protocol:

  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



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.




2.6 - ATR-FTIR spectroscopy assay



To complete our analysis on the protein, we studied the conformational changes which HpNikR goes under 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: 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.





2.7 - CryoTEM microscopy of purified HpNikR


High quality of the CryoTEM image displays: Correct sample preparation Correct set of experimental and strumental conditions High probability that the image is relative to purified HpNikR protein. Assuming that ribosomal particles, whose molecular weight is around 200 kDa, have a diameter around 20 nm, the particle of diameter of around 1-2 nm which can be seen may coincide with a particle of MW around 20 kDa. HpNikR MW is around 17 kDa.

3 - Nickel responsive promoters characterization data


Fluorescence Decay assay


We set up these experiments 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 and the double divergent promoter, thus repressing the transcription of GFP. (Nickel and IPTG added per flask: 0.3 ul from stock 10 ug / ul; 5 ul 1M)



3.1 - pnikR


To characterize this part we used BBa_K1151036. These data shows the ability of HpNikR to regulate pnikR, regulating GFP expression.







3.2 - Double divergent promoter pnikR-pexbB


To characterize this part we used BBa_K1151038. These data shows the ability of HpNikR to regulate divergent promoter, regulating GFP expression.








All the graphs show 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, HpNikR (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 HpNiKR 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 the two typical wavelenghts of GFP in order to have a complete and secure framework of the experiments.

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