May

Project Design

The month of May was largely focused on the selection and design of our Biobrick parts and devices. Two weeks were dedicated to the selection process for our team's 2014 iGEM project. Each acting team member identified a problem and proposed a solution via a genetically engineered organism; through discussion and popular vote, it was eventually determined that our team would work to express functional sMMO in E.coli for the purpose of methane remediation.

At this time, the team also voted to continue characterization and expansion of the RBS insertion primers first designed by Georgia Tech's 2013 iGEM Team.

May saw very little bench work besides training modules to develop wet lab skills and build our supply of biological resources.

June

RBS and Promoter Primer Testing

Starting in June the team began testing of newly designed RBS insertion primers as well as newly designed promoter insertion primers based upon the same concept. Initial tests with these primers ultimately failed. It was deemed that the protocols and techniques developed by the team were not adequate enough for job. However, by the end of the month the team had achieved successes in inserting both a high and a low efficiency RBS site in front of the mCherry reporter gene in pSB1C3.

sMMO Project Planning

No bench work was performed on the sMMO project in the month of June, instead the time was spent meticulously designing our selected sMMO genes for DNA synthesis in order to ensure optimized expression in E. coli and an easy to manipulate gene in the lab. Additionally, the high cost of synthetic DNA drove our team to spend a great deal of time fundraising and obtaining discounts from molecular biology product suppliers.

July

RBS and Promoter Primer Function Verification

Come July, refinement of the techniques and protocols used for RBS and promoter insertion finally came to fruition. Breaking the protocol into separate steps for RBS insertion and promoter insertion as well as a re-designing of the original promoter primers led to the verification of three new RBS primers and two new promoter primers. Although the strength of these PCR inserted sites had not yet been checked by flow cytometry, sequencing showed that the sequences had been inserted as predicted.

sMMO Gene Arrival

The synthesized sMMO genes arrived during this month, and all efforts went towards inserting these genes into vectors (if they weren't already) and transforming E.coli with them for the purpose of creating safe frozen stocks.

August

RBS and Promoter Relative Expression Analysis

Finally Flow cytometry characterization of the RBS and promoter primers was initiated. The cytometry provided information on the relative expression rates of combinations of various strength RBS and promoters. This knowledge aided in our selection of primers to use with the individual sMMO genes.

It was noted that the value of these primers would be more tangible if they were also compared to an mCherry reporter with an RBS and promoter site synthesized through more traditional 3A assembly or ordered digestion and ligation.

Generation of New Biobrick Parts

sMMO genes Z, B and C were inserted into Biobrick format over the month of August in vector pSB1C3.

September

Expression Vector Creation

Over the course of September, the real work of inserting RBS and Promoter sequences into the sMMO pSB1C3 plasmids began. In this time RBS sites were added to the sMMO Z, B, and C genes. Additionally the promoters were also added to sMMO Z and B plasmids thus completing our production of the sMMO Z and B expression vectors.

Generation of New Biobrick Parts

sMMO gene Y was inserted into Biobrick format over the month of September in vector pSB1C3.

GSU Collaboration

Mambalgin-1 was biobricked using 5' Extension PCR in the pSB1C3 vector as the first step in the effort to express Mambalgin protein in E. coli.

October

GSU Collaboration

Biobricked Mambalgin-1 had regulatory RBS and Promoter sequences inserted via our insertion primer process. It has since been sent back to GSU and we have assisted in the characterization of Mambalgin-1 expression in E. coli.