Georgia State University

In a meeting with Georgia State University’s IGEM team, they informed us of their project to express the gene Mambalgin-1 in E.coli. Mambalgin is a powerful and non-addictive analgeasic isolated from black mamba venom; given it's eukaryotic origins, GSU has gone through the hoops to create a single functional Yeast expression vector for Mambalgin. In this year's project they aimed to both optimize and characterize the production of Mambalgin in E.coli. It was evident that they planned to spend much of the summer creating new varied strength expression vectors for Mambalgin. Having already seen promising results from our new insertion primers, we informed them that, using our revolutionary RBS and Promoter Primer PCR, we could add a ribosome binding site and a promoter to the Mambalgin gene both cheaper and faster. Their interest in our proposal thus led to the creation of a new RBS insertion primer specifically for their sequence.

First, the gene was put in Biobrick format using 5’ Extension PCR, using the two primers shown below to add the Biobrick prefix and suffix to either side of the gene.

• Forward Primer sequence(Adds Biobrick Prefix)

  

• Reverse Primer sequence(Adds Biobrick Suffix)

  

• PCR Product using the above two primers with the Mambalgin-1 template DNA

  

Together the primers amplified a 225 bp long segment of DNA as predicted. This PCR product was subsequently ligated into a backbone and transformed into E.coli; it was later confirmed successful by sequencing. Next, the ribosome binding site was inserted using the RBS Primer PCR method that was developed here at Georgia Tech. Since the start codon in this case was 'CTG', a new RBS primer was designed with the same structure and components ( prefix, RBS, scar and start codon) as our universal primers, but with ‘CTG’ as the ending base pairs. Success of this PCR ultimately confirmed the adaptability of the RBS insertion method.

• RBS insertion primer sequence

  

• New PCR product using the RBS insertion primer and the Reverse Primer from above (only prefix end shown)

  

This PCR product was ligated into a backbone and transformed into E. coli again and confirmed with sequencing to be ready for Promoter Primer PCR. To add a promoter in front of the RBS, a promoter primer previously developed for our own project was used since it was compatible with the Prefix/RBS site.

• Final Mambalgin Sequence with regulatory RBS and Promoter regions inserted

  

This PCR product was ligated into a backbone and transformed into E.coli one last time and confirmed with sequencing. After all of this was accomplished, we sent a sample of this new plasmid to GSU and assisted them in the protein characterization process. The collaboration effectively created mambalgin expressing E.coli in a much more time and cost-effective manner than via traditional digestion and ligation or 3A assembly. Our RBS and Promoter PCR Primer Design and PCR eliminate the need to buy constructed sequences and the success of insertion can be confirmed quickly via gel electrophoresis.

Lambert Highschool

During the summer the Georgia Tech iGEM team aided in critiquing the Lambert High School iGEM team’s presentation for the manufacture of chitosan, which helps preserve fruit and vegetables from spoiling rapidly. On two different occasions, our research team oversaw the Lambert iGEM team’s presentation, helping them improve their presentation format, style, and slide orientation.

Our Team's Wiki Czar also contributed to the design, formatting, and HTML coding of Lambert High School's Wiki. Just three days before the iGEM 2014 High School Wiki freeze, Lambert High School's wiki was difficult navigate and with formatting and a color palette liable to give the reader a headache if stared at for too long. All of this was alleviated over the next 3 days as our Wiki Czar and the Lambert Team coded together to create a wiki that really captured the value of their work and that they could proudly display at their own iGEM Jamboree.

University of Virginia

During the summer, two of our team members attended an iGEM meet-up event hosted by the 2014 UVA iGEM team to discuss human practices. Several different teams from the southeast attended the event. At the meeting, the possibility of creating a synthetic biology course was brought up with the goal of introducing basic synthetic biology knowledge to a wider audience.

After the event, UVA created a potential outline for the online synthetic biology course. We took these ideas and designed a few lesson plans for the course and we created two powerpoint presentations to be the basis for course lessons on plasmids and Biobrick parts. Using the powerpoint presentations, we practiced presenting the lesson material with a small student body audience (around 20 people). The audience consisted of students interested in synthetic biology and getting involved with iGEM in the future. Moving forward, we are planning on refining these lessons, writing several more lesson plans on subjects including PCR, gel electrophoresis, transformations, restriction enzymes, promoters and primers, and 3A assembly, and creating pencasts/videos to bring this knowledge to a wider audience to include people that might not even be students.

Powerpoint for Plasmids Presentation

Powerpoint for Biobricks Presentation