Bronze badge

- Registered for iGEM;
- Completed the Judging form;
- Created and shared a description of our team's project using the iGEM wiki
- We documented and submited in Registry 9 new parts (BBa_K1819000, BBa_K1819001, BBa_K1819002, BBa_K1819003, BBa_K1819004, BBa_K1819005, BBa_S05291, BBa_S05307, BBa_S05308)
- We have a wiki;
- We have stated clear Attributions;
- We have prepared a poster and a talk to the iGEM Jamboree.

Silver badge

- We experimentally validated four new BioBrick Parts of our own design and construction worked as expected.
- We Documented the characterization of this part in the Main Page section of the Registry entry for the Parts:
BBa_K1819006
BBa_K1819007
BBa_K1819008
BBa_K1819009
- These parts are different from those documented in Bronze medal criterion #6
- Submissions adhered to the iGEM Registry guidelines
- We organized a Latin America teams meeting, presented our project in academic events, visited tire pyrolysis and tire recycling companies, performed a research of Women in Science, projected our patent and talked to innovation specialists, and made an educational activity with children. All Human Practices is described in details in our Practices pages.

Gold badge

To check the viability of our kill switch we planned two tests, using different promoters, all induced by addiction of a sugar (arabinose, rhamnose and IPTG) in the culture medium.

The first test monitors the production of two fluorescent proteins, GFP and RFP (Red Fluorescent Protein). The idea is that in the presence of the inductor (arabinose, rhamnose or IPTG) the bacteria produces GFP and tetracycline, which will inhibits the production of RFP and in the absence of the inductor the production of GFP and tetracycline would cease and the production of RFP would get higher. The assemblies are shown below.

The second test follows the same principle but instead of two fluorescent proteins we used GFP only. In this case, GFP would be produced if the E. coli grew in a medium lacking the inductor. The assemblies are shown below.

Parts design

Latex Clearing Protein (Lcp) and Rubber Oxygenase A (RoxA)

We received Lcp and RoxA sequences optimized for E. coli cloned in pUC9 from Prof. Dr. Dieter Jendrossek and the first thing we did was check their biobrick compatibility searching for EcoRI, PstI, SpeI and XbaI sites. Lcp didn’t possess any of these sites but we found an EcoRI site between bases 207 and 211 of RoxA sequence. To solve this problem we planned a set of primers changing a thymine for a cytosine, keeping the asparagine codon, to perform site directed mutagenesis.



To amplify these sequences and clone in pSB1C3 we design the primers in a way that they would contain the biobrick prefix and suffix as well as NdeI and SacI sites for protein fusion, as mentioned previously.
The primers for Lcp are the following.



The primers for RoxA are the following.



The meaning of the colors is in the table below.

Other parts

Some of the parts we needed for the main assembly and exportation test were synthesized as gBlocks® Gene Fragments by Integrated DNA Technologies (IDT). One of them was the signal TAT, in addition to its sequence this part had the prefix and suffix used in biobricks as well as a SacI site. The sequence is shown below.

SignalTAT

Another part we decided to synthesize was the GFP used in the exportation tests. In this case we had to change a thymine for a cytosine in the blue area from 282 to 284 bases to eliminate an undesirable NdeI site. Another modification was a linker (represented in grey in the sequence below) add so the GFP wouldn’t interfere with Lcp and RoxA folding.

References

(1) Yikmis, M; Arenskötter M, Rose K, Lange N, Wernsmann H, Wiefel L, Steinbüchel A. “Secretion and transcriptional regulation of the latex-clearing protein, Lcp, by the rubber-degrading bacterium Streptomyces sp. strain K30”; Appl Environ Microbiol. 74(17):5373-82., September 2008.
(2) “OmpA outer membrane protein A fused to linker; displays proteins on cell surface”, available on: http://parts.igem.org/Part:BBa_K103006
(3) Poulsen, L. K.; Larsen, N. M.; Molin, S.; Andersson, P; “A family of genes encoding a cell-killing function may be conserved in all Gram-negative bacteria”; Molecular Microbiology, 3(11), 1463-1472, 1989.