Difference between revisions of "Team:Tec-Chihuahua/Results"

While we were constructing our BioBricks™, we realized there was a problem because the insert wasn't ligating to pSB1C3. Everything seemed correct in the transformation protocol, but once the DNA was visualized on an agarose gel our insert was missing and only pSB1C3 was evident. We then thought there was a problem during ligation. Therefore, we analyzed IDT’s synthesized sequence and identified that there were missing additional base pairs that flanked the restriction site so that the enzyme could properly cling and cut the sequence.

This issue was troubleshot with the TecCEM iGEM team. They had the great idea to create primers that add six base pairs before the prefix and after the suffix thereupon amplifying the gene of interest. This collaboration was crucial for the construction of our BioBricks. Read the entire collaboration

The three composites of our creation, apidaecin - [BBa_K2834003], defensin 1 - [BBa_K2834005], and abaecin - [BBa_K2834006] were characterized with the intention of expressing the peptides in E. coli BL21 (DE3) by IPTG induction. Its antimicrobial activity was evaluated against Gram-positive bacteria with antibiotic susceptibility testing by measuring OD₆₀₀ in broth.

The three composites were synthesized by IDT® with the prefix and suffix flanking the region of interest. The final parts resulted in a sequence of 310 bp for apidaecin, 415 bp for defensin 1, and 361 bp for abaecin. Once the synthesis arrived, and the PCR was carried out, double digestion with EcoRI-HF and PstI restriction enzymes was made to each composite, and the chloramphenicol linearized plasmid backbone (pSB1C3) for following ligation of the backbone with each one of the fragments. This resulted in complete expression plasmids of 2337 bp for apidaecin, 2442 bp for defensin 1, and 2388 bp for abaecin. Afterward, Escherichia coli BL21(DE3) cultures were transformed by heat shock for following antibiotic selection of clones. Next step consisted of plasmid extraction and electrophoresis of the undigested plasmids, linearized plasmids with one enzyme, and double digested plasmids. Agarose gels allowed the confirmation of the correct plasmid constructions.

Following the construction of each BioBrick, it was necessary to induce protein production. Since the T7 promoter regulates transcription of the construct, isopropyl β-D-1 thiogalactopyranoside (IPTG) is used as an inducer for T7 RNA polymerase production. The concentration of IPTG used was 0.5 mM. After induction, the cultures were incubated for six hours at 37 °C and 225 rpm. After that, protein extraction by lysis solution was made in order to obtain the soluble peptides. For insoluble peptides, the sample was treated with lysis solution + 6 M urea.

After protein extraction, electrophoresis in polyacrylamide gel (12%) was performed to corroborate the peptides of interest were indeed expressed. To calculate the molecular weight of each peptide, the Promega Biomath Calculator was used.

Bands of the following kDa were expected

The iGEM experience taught us a lot of unique things, and as we had significant achievements, we went through many unexpected obstacles. Throughout the year we faced many challenges for finally reaching our goal; we believe that the effort was worthwhile.