Team:Alverno CA/Design

Design

The goal of of our project is to counteract a problem in synthetic biology known as supercoiling. Supercoiling is a possible consequence that can occur in the construction of a multi-genetic circuit. When the two genes transcribe, the double helix is opened in order to allow for the expression for the genes, however, the dna in between the two genes receive extraordinary amounts of tension when both genes try to transcribe at the same time, which can lead to the over transcription of one gene, and the under expression of the other gene. This can greatly affect the predictability of gene expression.

In order to solve this problem, we have assembled a circuit in which RFP and GFP are constructed in the same plasmid. To test if we could reduce the supercoiling between these two genes, we designed several different strategies that we felt may alleviate the problem. The first method we tried was adding random base pair sequences in between the two genes, with lengths ranging between fifty and one thousand and five hundred base pairs. We created this in the hope that the space in between the two genes would alleviate the tension that causes the supercoils. Another of our methods was to use a dCas9 clamp on the space between the RFP and GFP to hopefully clamp down on the supercoiling. We utilized Golden Gate Assembly in order easily build several variations of these plasmids by cloning with several different parts.