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 the Dalton High School iGEM team:
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 the John Hopkins Software Team:
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.
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.
We are registered!
Can be found here.
Where do you think you are right now?
See you in Pittsburgh!
See our list of submitted parts.
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