Team:Ciencias-UNAM/Achievements

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Achievements

We characterised LL-37 antimicrobial peptide from Team Trieste 2008. They used Lac promoter to express this peptide, hoping to inhibit cell growth. They did not have positive results, as the control experiment growth the same way as the one with LL-37 expression with IPTG. We approached this problem with two solutions. The first problem we saw in Trieste's characterisation was that Lac promoter has a very high basal level of transcription. Given pSB1C3 has a high copy number, it is transcribing a high amount of LL-37, still with no IPTG in the media. This could mean that there is selection pressure on E. coli to loose the plasmid, which could be one of the reasons the did not see changes between the control cells and the cells under IPTG induction. We addressed this problem by changing the promoter, constructing device BBa_K1230007. This device is under the pBad promoter, which works with arabinose. This promoter has a basal level of almost 0, so we are sure that it is not transcribing any LL-37 that could be making the cells to loose the plasmid. The second problem we addressed was the change in the codon usage they used. By changing the codons to produce more peptide in E. coli, the peptide may not have sufficient time to fold properly, altering it's function. We solved this problem by using the normal codon usage of this peptide, allowing it to fold properly into it's final conformation.

The principal aim of our project was to collaborate as much as possible to the iGEM mission to develop science freely. The parts we designed followed this principle, by being useful in very aspects, apart of our own project. We developed a Multiple Antibiotic Resistance protein, which acts as a transcriptional activator in E. coli, it allows the expression of the acrAB and tolC operons, which activate the AcrAB-TolC efflux pump, a mechanism that has been related with resistance to organic solvents, dyes, detergents, antibiotics such as chloramphenicol, tetracycline, novobiocin, erythromycin, fusidic acid and cloxacillin, as well as to cationic antimicrobial peptides, such as LL-37, HNP-2 and HBD-1. This part can be used in several ways in different iGEM projects. We also sent to the parts registry the LL-37 peptide, with adequate codon usage, an improvement on part BBa_K875000, that allows LL-37 expression.

As part of our collection, we also designed the device BBa_K1230009, which is the promoter as well as the coding sequence for the repressor AraC which is transcribed in the opposite direction, with the Elowitz repressilator. This part will allow an easy way to produce proteins by Arabinose induction, improving the way characterization is made on iGEM.
Requirements for a Bronze Medal:
Register the team, have a great summer, and plan to have fun at the Regional Jamboree.
Successfully complete and submit this iGEM 2013 Judging form.
Create and share a Description of the team's project using the iGEM wiki and the team's parts using the Registry of Standard Biological Parts.
Plan to present a Poster and Talk at the iGEM Jamboree.
Document at least one new standard BioBrick Part or Device used in your project/central to your project and submit this part to the iGEM Registry (submissions must adhere to the iGEM Registry guidelines).
https://2013.igem.org/Team:Ciencias-UNAM/Project/WetLab/BioBricks

Additional Requirements for a Silver Medal:
Experimentally validate that at least one new BioBrick Part or Device of your own design and construction works as expected.
Document the characterization of this part in the 'Main Page' of that Part's/Device's Registry entry
Submit this new part to the iGEM Parts Registry (submissions must adhere to the iGEM Registry guidelines)
Part Number(s): BBa_K1230004, “MarA Generator”
http://parts.igem.org/wiki/index.php?title=Part:BBa_K1230004
Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe one or more ways in which these or other broader implications have been taken into consideration in the design and execution of your project.

Additional Requirements for a Gold Medal:
Improve the function of an existing BioBrick Part or Device (created by another team or your own institution in a previous year), enter this information in the 'Experience' section of that Parts's Registry entry), create a new registry page for the improved part, and submit this part to the iGEM Registry (submissions must adhere to the iGEM Registry guidelines).
Part Number(s): BBa_K1230007, “araC-pBad-rbs-LL37”
http://parts.igem.org/wiki/index.php?title=Part:BBa_K1230007
Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation.
We did formal Petition to Mexican Congress to review DNA import/export politics, addressing a letter to Congressman Rúben Benjamín Félix Hays, President of the Science and Technology Commission of the Mexican Congress, given that in Mexico the Federal Commission for Protection against Health Risks (COFEPRIS), regulates, with a lot of normative has flaws, both imports and export of genetic material for research. These flaws make genetic material imports unavailable for very large periods of time. Noting the urge of creating a law initiative in this matter and proposing changes in the already existing norms, we take in account aspects such as security, ethics, and above all, the aspect of sharing.
Best BioBrick Measurement Approach:
Our experiments focused on the measurements of the expected phenotypes, which in our case, involve the increase or decrease of viability or inhibition. To measure the experiment in this way is relevant, since the expected function of the part is a phenotype. Although measuring via reporters is useful to infere the production of a certain protein, the cuatitiative measure of the effect is more relevant in parts like ours. The designed experiments allow to use two variables (in this case the induction with IPTG and the concentration of antibiotic) and to show not only the characterization of a promoter (as it is often done) but also a cuantitative measure of the desired phenotype.
Best Model: We were able to recognize parameters based on the system dynamics.
Team_Parts
Best new BioBrick Part, Natural:
Part Number(s): BBa_K1230000, “AcrAB-TolC multidrug efflux transport system activator from E. coli”
http://parts.igem.org/wiki/index.php?title=Part:BBa_K1230000
Best new BioBrick Part or Device, Engineered:
Part Number(s): BBa_K1230004, “MarA Generator”
http://parts.igem.org/wiki/index.php?title=Part:BBa_K1230004
Most Improved Registry Part:

Part Number(s): BBa_K1230007, “araC-pBad-rbs-LL37”

Short description of improvements made to the part:

We characterised LL-37 antimicrobial peptide from Team Trieste 2008. They used Lac promoter to express this peptide, hoping to inhibit cell growth. They did not have positive results, as the control experiment growth the same way as the one with LL-37 expression with IPTG. We approached this problem with two solutions. The first problem we saw in Trieste's characterisation was that Lac promoter has a very high basal level of transcription. Given pSB1C3 has a high copy number, it is transcribing a high amount of LL-37, still with no IPTG in the media. This could mean that there is selection pressure on E. coli to loose the plasmid, which could be one of the reasons the did not see changes between the control cells and the cells under IPTG induction. We addressed this problem by changing the promoter, constructing device BBa_K1230007. This device is under the pBad promoter, which works with arabinose. This promoter has a basal level of almost 0, so we are sure that it is not transcribing any LL-37 that could be making the cells to loose the plasmid. The second problem we addressed was the change in the codon usage they used. By changing the codons to produce more peptide in E. coli, the peptide may not have sufficient time to fold properly, altering it's function. We solved this problem by using the normal codon usage of this peptide, allowing it to fold properly into it's final conformation.

Best Part Collection: (give the range of part numbers)

Part Number(s): BBa_K1230000, BBa_K1230001, BBa_K1230004, BBa_K1230007, BBa_K1230009, BBa_K1230010, BBa_K1230011, BBa_K1230013, BBa_K1230015, BBa_K12300016 not found

Short description of collection function:

As part of our collection, we also designed the device BBa_K1230009, which is the promoter as well as the coding sequence for the repressor AraC which is transcribed in the opposite direction, with the Elowitz repressilator. This part will allow an easy way to produce proteins by Arabinose induction, improving the way characterization is made on iGEM.