We have modified pSB1C3, the standard iGEM shipping plasmid, to allow for easy conversion of our yeast Golden Gate (yGG) parts to the BioBrick standard. We submitted three modified pSB1C3 plasmids, one to convert each type of yGG part: promoters, open reading frames (ORFs), and terminators. Being able to convert parts between standards represents a major challenge, in large part because standards are constantly changing. Thus, we feel this set of 'conversion vectors' is an important contribution.
Collaboration with: Dalton High School iGEM 2012.
A former PhD student at Johns Hopkins University and a current high school science teacher and iGEM advison at the Dalton School in New York City, Dr. Jennifer Hackett approached the Hopkins wetware iGEM team to discuss a mutual interest in characterizing yeast promoters. As a result of this conversation, we suggested that as one component of the Dalton School's iGEM project, Dr. Hackett could implement both our Parts Course as well as our Yeast Transcriptional Unit Assembly Standard (RFC88). In brief, we suggested that the Dalton team could subclone a series of yeast promoters, flanked by BsaI sites and our signature overhang sequences, so they could efficiently and directionally assembly these promoters upstream of genes encoding different fluorescent proteins. As part of this collaboration, we selected ~30 yeast promoter sequences for the Dalton team to clone. Promoters were chosen if they could be induced (e.g. by temperature or sugar source), or would drive expression of the downstream fluorescent protein with varying strengths. The Dalton School had a successful project and were able to clone almost the entire set of promoters. They are now working on assembling transcriptional units and testing the effects of the different promoters.
Collaboration with: JHU Software iGEM 2012.
The second of our collaborations was with our colleagues on the Johns Hopkins software team. They developed AutoGene, "an all-encompassing plasmid design suite meant to streamline the process of both annotating and building sequences." We sent them several plasmid sequences and they returned the annotated plasmids. We then provided feedback to the software team on the performance of AutoGene. This collaboration was beneficial to the JHU Software team as our requests and user feedback helped them to develop AutoGene.
We designed a synthetic biology lab manual to be used in introductory biology lab courses. The Yeast Golden Gate Lab Course.
We showed that our ethanol-induced promoters showed different responses to ethanol. We showed that our CYP2E1 expressing yeast strains lowered the concentration of ethanol over a 24 hour fermentation. We showed that our Golden Gate to BioBrick conversion vectors could easily convert our Golden Gate promoters, coding sequences, and terminators to the BioBrick standard for submission to the Registry.
We characterized the operation of our ethanol-induced promoters by using them to express GFP under different ethanol concentrations. We measured the effect of CYP2E1 expression under ethanol-induced and constitutive promoters on the ethanol concentration during fermentation.
Our team has been registered for the Igem competition.
The judging form has been completed. It details that we believe we deserve to win the Gold medal due to the reasons specified therein. It also shows our best new parts. Judging form can be found here
The wiki, through the intensive and dedicated work of our wiki master, James, has been set up and completed. It includes our project and our new assembly standard, Golden Gate.
Our poster has been designed and is ready for the flight to Pittsburgh. Our presentation is set to be a stellar one.
Our ethanol inducible promoters: BBa_K799001, BBa_K799003, BBa_K799005, BBa_K799007, BBa_K799009 were placed in control of GFP and fluorescence data was analyzed at various ethanol concentrations. The graphs can be seen on the respective page of each part. See our list of submitted parts.
"
