Team:UC Chile2/Bactomithril/Notebook

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==December 2011==
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<a href="https://2012.igem.org/Team:UC_Chile2/A_brief_summary_on_december">Here</a>  you can find out what did in December. (e.g. the course on Synthetic Biology)
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==March, 5 - 11, 2012 ==
==March, 5 - 11, 2012 ==
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Also, this week we tried to redesign our project. Our first task is to continue with our attempts to make a biobrick with the spider silk monomer ADF-3. We ordered the primers that we need to do that.
Also, this week we tried to redesign our project. Our first task is to continue with our attempts to make a biobrick with the spider silk monomer ADF-3. We ordered the primers that we need to do that.
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Now we are exploring the possibility of improve the product of the secretion system developed by Christopher Voig in his paper “Engineering the Salmonella type III secretion system to export spider silk monomers”. One idea is to attach a growth factor (e.g. recombinant human BMP-2) to the molecule of ADF-3.
Now we are exploring the possibility of improve the product of the secretion system developed by Christopher Voig in his paper “Engineering the Salmonella type III secretion system to export spider silk monomers”. One idea is to attach a growth factor (e.g. recombinant human BMP-2) to the molecule of ADF-3.
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But the problem with the secretion system developed by Christopher Voig is that it works on salmonella, and we cannot work with such an infectious bacteria.  
But the problem with the secretion system developed by Christopher Voig is that it works on salmonella, and we cannot work with such an infectious bacteria.  
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Furthermore, we studied carefully the paper “Engineering the Salmonella type III secretion system to export spider silk monomers” and its supplementary information. We realized that each protein secreted with this system comes with a 3x FLAG (DYKDDDDK)3. It is a priority for us to find if someone can share us a little of the anti-FLAG antibody (Sigma Cat# F3165). That way, if we use this secretion system we will be able to quantify the production of our protein of interest.
Furthermore, we studied carefully the paper “Engineering the Salmonella type III secretion system to export spider silk monomers” and its supplementary information. We realized that each protein secreted with this system comes with a 3x FLAG (DYKDDDDK)3. It is a priority for us to find if someone can share us a little of the anti-FLAG antibody (Sigma Cat# F3165). That way, if we use this secretion system we will be able to quantify the production of our protein of interest.
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But, if we want to use Voigt´s system, we need attenuated salmonellas with their pathogenicity island 1 intact.  Something similar to Salmonella Typhimurium SL1344. After some research we found a professor of our University that works with salmonella (Dr. Susan Bueno). But she was out of the country and we could only send her some emails asking for the salmonella.   
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But, if we want to use Voigt´s system, we need attenuated salmonellas with their pathogenicity island 1 intact.   
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Something similar to Salmonella Typhimurium SL1344. After some research we found a professor of our University that works with salmonella (Dr. Susan Bueno). But she was out of the country and we could only send her some emails asking for the salmonella.   
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Finally the primers necessary for biobricking ADF-3 arrived!
Finally the primers necessary for biobricking ADF-3 arrived!
And we have got other important material: we talked with Dr. Hugo Olguin, and he is willing to share with us his anti-FLAG antibody. Furthermore, he allowed us to do the western blot in his laboratory.
And we have got other important material: we talked with Dr. Hugo Olguin, and he is willing to share with us his anti-FLAG antibody. Furthermore, he allowed us to do the western blot in his laboratory.
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Additionally, we achieved significant advances in the wiki, especially in the section Human Practices.  
Additionally, we achieved significant advances in the wiki, especially in the section Human Practices.  
Unfortunately, we still lack of salmonella.
Unfortunately, we still lack of salmonella.
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After some research we found the promising projects of the Japanese iGEM team HokkaidoU 2010 and 2011. They used the E. Coli (k-12) strain SGSC4024, from Salmonella Genetic Stock Center(SGSC) in University of Calgary, Canada  (http://people.ucalgary.ca/~kesander/index.html). This strain carries a pBeloBAC11 vector encoding a genome fragment of Salmonella enterica serovar Typhimurium LT2 which covers the SPI-2 region [B_STM07H21 SGSC4024 1464540～1562427]
After some research we found the promising projects of the Japanese iGEM team HokkaidoU 2010 and 2011. They used the E. Coli (k-12) strain SGSC4024, from Salmonella Genetic Stock Center(SGSC) in University of Calgary, Canada  (http://people.ucalgary.ca/~kesander/index.html). This strain carries a pBeloBAC11 vector encoding a genome fragment of Salmonella enterica serovar Typhimurium LT2 which covers the SPI-2 region [B_STM07H21 SGSC4024 1464540～1562427]
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We asked the iGEM team members of HokkaidoU 2011 if the E. Coli (k-12) strain SGSC4024 is capable of secreting proteins to the external environment. But unfortunately it seems that is only capable of injecting molecules into other cells, and that is not what we need.
We asked the iGEM team members of HokkaidoU 2011 if the E. Coli (k-12) strain SGSC4024 is capable of secreting proteins to the external environment. But unfortunately it seems that is only capable of injecting molecules into other cells, and that is not what we need.
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==July 30 – August 5, 2012 ==
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In the lab we did PCR to amplify ADF-3 and ADF-3+ (i.e. ADF-3 with the tags included in the plasmid that Christopher Voigt sent to us). Unfortunately the PCR did not work.
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The next day we tried to amplify the backbone pSB1C3, ADF-3+ and ADF-3 with touch-down PCR. The two first amplicons seemed to be all right. Nevertheless, later we purificated and digested the DNA to verificate if the amplicons corresponded to our parts, but the size sequences resulted to be incoherent. We have to try again.
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==July 30 – August 5, 2012==
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This week we did PCR of the backbone pSB1C3, ADF-3+ and ADF-3 again. It failed again. Are our primers wrong?
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However, we persisted tirelessly with another PCR of pSB1C3, ADF-3+ and ADF-3, this time with a Tm=59º C. It worked for ADF-3+!!
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We purified the DNA sequence of ADF-3+. Afterwards we did PCR again, but this time to amplify the backbone of ADF-3 that has the promotor pBAD, and to amplify the vector backbone for ADF-3+.
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The PCR of the backbone of ADF-3 that has the promotor pBAD looks successful! Later we proceeded with the corresponding DNA purification.
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Joyfully we proceeded with the Gibson Assembly to join ADF-3 to its backbone with pBAD. We transformed the E. coli, but no colony survived.
==August 6 – 12, 2012==
==August 6 – 12, 2012==
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We did another PCR, but this time amplified ADF-3 with a Tm=56º C ;  The vector backbones of ADF-3+ and ADF-3 with Tm=60º C and touch-down PCR of the vector backbones of ADF-3+ and ADF-3 with T annealing = 54º C.
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Tragically, none of them worked.
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The next day we did PCR again with Tm=59ºC of ADF-3 and the vector backbones of ADF-3+ and ADF-3.
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The PCR of ADF-3 was successful! Later we did the purification of the DNA.
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Afterwards, we completed the Gibson Assambly of ADF3 with the backbone PSB1C3 and the transformations of the E. coli.
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The next day finally we had some transformed E. coli! Immediately we proceeded with colony PCR to verify if the transformed bacteria correspond to our designs. Unluckily, the colony PCR was negative (the DNA strand that we obtained had a smaller molecular weight than what we expected).
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So that we repeated the PCR for the vector backbones of ADF-3 and ADF-3+. The bands were from the right length! Afterwards we proceeded with the Gibson Assembly of ADF-3 with its backbone, and of ADF-3+ with its backbone. Both Gibson Assemblies were positive! Finally we continued with a colony PCR.
==August 13 – 19, 2012==
==August 13 – 19, 2012==
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We purificated the DNA from the colony PCRs that were successful, and then digested it. Fortunately one digestion of ADF-3 and other of ADF-3 had the correct size.
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Later we did PCR to amplify the backbones of ADF-3 and ADF-3+ (both with pBAD). But we made some errors and added the wrong primers.
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The next day we repeated the PCR, and the backbone with pBAD of ADF-3 had the right size.
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Then we send for sequencing ADF-3 and ADF-3+. Later we realized that we sent the wrong sample of ADF-3!!
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We also transformed some E. coli with backbone with pBAD of ADF-3. The following day we did colony PCR to these E. coli, but we did not obtain the desired  results. Then we cultivated the E. coli transformed with the backbone with pBAD of ADF-3 in 200 mL of LB with 1nM of arabinose.
==August 20 – 26, 2012==
==August 20 – 26, 2012==
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We did colony PCR of 3 colonies transformed with the backbone with pBAD of ADF-3, but the results were negative.
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The next day we did colony PCR again, but this time with other method:
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1) Put 100 mL of bacteria in eppendorf tubes of  2µl and heat at 95ºC for 10 minutes.
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2) Centrifuge at maximum speed for 5 minutes.
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3) Do Colony PCR with the supernatant as the template.
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Unfortunately, the results were negative.
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And we became the results from the sequencing. ADF-3 is incorrect (as we expected, because we sent the wrong sequence), and ADF-3+ is a bit weird. Bernardo never trusted the PCR band of this sequence anyway…
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The next day we sent for sequencing ADF-3 (this time the right sequence). Some days later we became the results from this sequencing, and it were negative. This is terrible! 
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Finally, we prepared the materials for the community outreach activity for Penta UC, the Talents Development and Study Program at Catholic University. And we participated in this activity. More information in Human Practices/Community
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Outreach.
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==August 27 – September 3, 2012==
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We transformed pBAD_ADF-3 in pSB1K3, and pBAD_ADF-3+ in pSB1K3.
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The next day we did colony PCR of those transformed bacteria at a Tm=59ºC. From all the colonies that we used, only one of pBAD_ADF-3 in pSB1K3 and one of pBAD_ADF-3+ in pSB1K3 had the correct size. Then we left these bacteria growing, so that we could purificate their DNA later .
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Latest revision as of 23:53, 21 September 2012

Project: Luxilla - Pontificia Universidad Católica de Chile, iGEM 2012