This year is the 2nd half of our two-year project - the creation of a DDx biosensor (you can read about earlier efforts on last year's Wiki). We wanted to address the pollution in the nearby Pine River - a river that runs next to our campus, and downstream to St. Louis, Michigan, a neighboring town. The floodplains of the Pine River in St. Louis has been contaminated for decades with DDT and it's derivatives (such as DDE, and other molecules collectively referred to as DDx). These molecules can persist in the enviroment and the soil for long periods of time, accumulating in some species and posing problems to the ecosystem and human health thanks to biomagnification. Our plan is to create a biosensor to enable the rapid and affordable detection of these molecules, aiding cleanup efforts both here in Michigan and worldwide. Making detection easier and cheaper can help direct funding and cleanup efforts in the most effective way possible, and can help restore trust in the local community.
One of the first things that the Alma iGEM team worked on this season was an improvement to the protocols for preparing the standard plasmid backbone. There were times were transformations weren’t working when they had in the past, and results weren’t turning out quite as expected based on previous experiments. Upon consultation with a few advisors and comparisons of notes with the times of solutions being prepared, we had reason to believe that the issue could be with our backbones that were being used. As such, research into iGEM teams that may have also had this problem yielded the work done by University of Chicago in 2019 with their ‘CUT’ primers. These improved the amplification of the pSB1C3 standardized iGEM backbone. While we did not conduct quantifiable testing on the level of success that these new plasmids yielded, they were noticeable in our lab procedures. The Alma iGEM would recommend any interested iGEM teams to consider utilizing these CUT primers in the future for production of pSB1C3 backbones.
The first major step in developing our project was that of deciding what would be utilized for the detection aspect of our biosensor. DDT is classified as a xenoestrogen, meaning that it serves as an agonist for estrogen in the bodies of most invertebrates. This made estrogen receptors a prime candidate for the receptor we would utilize to detect DDT. We then had to consider what estrogen receptors would be used, with fish, birds, primates, and potentially human being the best candidates that were found. This was based on research done by the team into what organisms were most affected by DDT. After working on codon harmonizing using web based tools, our choices were narrowed down to the California Condor, American Rainbow Trout, and Human estrogen receptors.
The team designed these parts and ordered them from companies affiliated with iGEM such as Integrated DNA Technologies (IDT) and Twist Bioscience. This step was not without its difficulties however; there was a considerable amount of time lost waiting for these parts to be assembled and delivered from these companies. There were a lot of delays and difficulties with the manufacturing process as a result of the genetic sequences being used. There was the concern of primer dimers within the sequences, and self annealing during the production of our sequences that resulted in some of our DNA being separated into 12 different pieces during the delivery process. It is worth noting for iGEM teams interested in utilizing companies to obtain genetic sequences, that there may be considerable delays and difficulties that can result as a consequence of the considerable complexity found in the production of eukaryotic DNA.
Following the delivery of these pieces of our biological circuit we began our work of assembling them into pSB1C3 and then transforming them into DH5A strains of E. Coli bacteria. There were some considerable roadblocks met with the assembly of the human estrogen receptor, and it is at this point believed to be producing a toxic byproduct that prohibits E. Coli from successfully utilizing the estrogen receptor and forming colonies. After about a month and a half of lab time however, successful completion of the transformation of each of the estrogen receptors was confirmed by colony gel electrophoresis and subsequent Sanger Sequencing performed by Michigan State University’s lab in Lansing, Michigan.
The next step was to then insert a promoter into the coding region for the estrogen receptor, as the pSB1C3 plasmid coding region contained the needed estrogen receptor, but was as of yet unable to express it. That proved itself to be immensely difficult and resulted in Alma iGEM eventually reordering the estrogen receptors with the promoters K608003, and K525998 present already connected to the estrogen receptor coding region. This has worked well, and we are currently awaiting sequencing results on the samples sent off to confirm our successful assembly of this part.
Something that was noticed during our team’s work over the summer was that there were EcoRI restriction enzyme sites at two separate locations about a thousand base pairs into our rainbow trout estrogen receptor coding region. As a team we designed primers that would then bridge the gap with imperfect primer binding sites, and remove the EcoRI restriction enzyme recognition sites. This was successful upon its first use in our thermocyclers. While not necessarily required by the rules of iGEM, we felt that it was necessary to do to insure that the parts being utilized and uploaded to the registry were fully legal and followed all rules for parts laid out by the organization.