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Team:Trieste/notebook4
From 2012.igem.org
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<div id="suicide" class="notebook_section"> | <div id="suicide" class="notebook_section"> | ||
<h2 class="notebook_title">Suicide System</h2> | <h2 class="notebook_title">Suicide System</h2> | ||
| - | + | Due to the negative results of the previous week , we decided to use another toxin from the registry, that we have already extracted from iGEM kit and amplified it through the transformation of DH5-alpha competent cells: Tse2 toxin (BB_K314200).<br/> | |
| + | |||
| + | We began to create our toxin construct, cutting Tse2 Toxin (EcoRI/SpeI) to clone it inside the double terminator BB_B0015, previously cut with EcoRI/XbaI.<br/> | ||
| + | |||
| + | Unfortunately we did not obtain a positive colony, so we tried different cloning approaches. | ||
</div> | </div> | ||
<div id="antibody" class="notebook_section"> | <div id="antibody" class="notebook_section"> | ||
<h2 class="notebook_title">Antibody</h2> | <h2 class="notebook_title">Antibody</h2> | ||
| - | + | We replaced pelB leader sequence with LPP-OmpA into the SIP fragment in order to attach the SIP on the bacterial surface. Then we cloned it downstream TLacO promoter. This construct was also tested in W3110 with p-REP 4. We induced its expression with IPTG (1mM). Following the same protocol, and we analysed the induced culture by Western blotting. Our fusion protein is being expressed. | |
</div> | </div> | ||
<div id="chassis" class="notebook_section"> | <div id="chassis" class="notebook_section"> | ||
| - | <h2 class="notebook_title"> | + | <h2 class="notebook_title">Cumate-Switch Regulation</h2> |
| - | + | <u><b>CymR</b></u> | |
| + | <br/> | ||
| + | We tried to transform again the same ligation and this time we get two positive clones. | ||
| + | First we made also a control-cut with EcoRI to exclude errors in the plasmid sequence. Then we cloned the CymR - B0015 fragment in the plasmid J61002 downstream the J23100 promoter. | ||
| + | We got no positive clones with the colony pcr so we tried two different kind of ligation: | ||
| + | <br/> | ||
| + | - the same as before but with the addition of SAP (shrimp-alkaline phosphatase) on the J61002 - J23100 after the digestion. | ||
| + | <br/> | ||
| + | - a ligation without gel purification and then we selected the non-red colonies. | ||
| + | <br/> | ||
| + | At the end we obtained some positives from the first procedure. | ||
| + | <br/> | ||
| + | <br/> | ||
| + | <u><b>T5 PROMOTER - CUMATE OPERATOR</b></u> | ||
| + | <br/> | ||
| + | We cloned in the two positive clones the E0240 (GFP) downstream the T5 promoter - Cumate operator. After the transformation we obtain no positive clones so we tried to repeat the same process just adding the SAP after the SpeI/Pst cut. | ||
| + | <br/> | ||
| + | We tried also to make a ligation without purification but with no positive results. | ||
| + | <br/> | ||
| + | We cut the positive pcr clones with XbaI/PstI but analyzing them in the gel we saw three different bands in every single clone that we cannot identify. So we re-digested other positive clones. | ||
| + | |||
</div> | </div> | ||
</div> | </div> | ||
| Line 28: | Line 52: | ||
<li><a href="https://2012.igem.org/Team:Trieste/notebook2">Week 2</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/notebook2">Week 2</a></li> | ||
<li><a href="https://2012.igem.org/Team:Trieste/notebook3">Week 3</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/notebook3">Week 3</a></li> | ||
| - | <li><a href="https://2012.igem.org/Team:Trieste/notebook4">Week 4</a></li> | + | <li class="select" ><a href="https://2012.igem.org/Team:Trieste/notebook4">Week 4</a></li> |
<li><a href="https://2012.igem.org/Team:Trieste/notebook5">Week 5</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/notebook5">Week 5</a></li> | ||
<li><a href="https://2012.igem.org/Team:Trieste/notebook6">Week 6</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/notebook6">Week 6</a></li> | ||
| Line 37: | Line 61: | ||
<li><a href="https://2012.igem.org/Team:Trieste/notebook11">Week 11</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/notebook11">Week 11</a></li> | ||
<li><a href="https://2012.igem.org/Team:Trieste/notebook12">Week 12</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/notebook12">Week 12</a></li> | ||
| + | <li><a href="https://2012.igem.org/Team:Trieste/notebooklast">Post European-Jamboree</a></li> | ||
</ul> | </ul> | ||
<img src="https://static.igem.org/mediawiki/2012/b/b0/Team_trieste.jpg" alt="Team iGEM 2012" id="igem_team" /> | <img src="https://static.igem.org/mediawiki/2012/b/b0/Team_trieste.jpg" alt="Team iGEM 2012" id="igem_team" /> | ||
| Line 46: | Line 71: | ||
Follow us also: | Follow us also: | ||
<a href="https://www.facebook.com/IGEMUNITS?ref=ts" target="_blank"><img src="https://static.igem.org/mediawiki/2012/f/f4/Ico_fb.png" alt="Facebook" class="fb" /></a> | <a href="https://www.facebook.com/IGEMUNITS?ref=ts" target="_blank"><img src="https://static.igem.org/mediawiki/2012/f/f4/Ico_fb.png" alt="Facebook" class="fb" /></a> | ||
| - | <a href="https://twitter.com/ | + | <a href="https://twitter.com/iGEMTrieste" target="_blank"><img src="https://static.igem.org/mediawiki/2012/2/21/Ico_twitter.png" alt="twitter" class="tw" /></a> |
</div> | </div> | ||
</div> | </div> | ||
Latest revision as of 18:06, 26 October 2012
The Jolly JoCare
a safe probiotic platform for protein expression
Week 4
More
Suicide System
Due to the negative results of the previous week , we decided to use another toxin from the registry, that we have already extracted from iGEM kit and amplified it through the transformation of DH5-alpha competent cells: Tse2 toxin (BB_K314200).We began to create our toxin construct, cutting Tse2 Toxin (EcoRI/SpeI) to clone it inside the double terminator BB_B0015, previously cut with EcoRI/XbaI.
Unfortunately we did not obtain a positive colony, so we tried different cloning approaches.
Antibody
We replaced pelB leader sequence with LPP-OmpA into the SIP fragment in order to attach the SIP on the bacterial surface. Then we cloned it downstream TLacO promoter. This construct was also tested in W3110 with p-REP 4. We induced its expression with IPTG (1mM). Following the same protocol, and we analysed the induced culture by Western blotting. Our fusion protein is being expressed.Cumate-Switch Regulation
CymRWe tried to transform again the same ligation and this time we get two positive clones. First we made also a control-cut with EcoRI to exclude errors in the plasmid sequence. Then we cloned the CymR - B0015 fragment in the plasmid J61002 downstream the J23100 promoter. We got no positive clones with the colony pcr so we tried two different kind of ligation:
- the same as before but with the addition of SAP (shrimp-alkaline phosphatase) on the J61002 - J23100 after the digestion.
- a ligation without gel purification and then we selected the non-red colonies.
At the end we obtained some positives from the first procedure.
T5 PROMOTER - CUMATE OPERATOR
We cloned in the two positive clones the E0240 (GFP) downstream the T5 promoter - Cumate operator. After the transformation we obtain no positive clones so we tried to repeat the same process just adding the SAP after the SpeI/Pst cut.
We tried also to make a ligation without purification but with no positive results.
We cut the positive pcr clones with XbaI/PstI but analyzing them in the gel we saw three different bands in every single clone that we cannot identify. So we re-digested other positive clones.
