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.
To test if we could reduce the supercoiling between these two genes, we proposed 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 of 500bp or 1000bp. We created this in the hope that the spacer in between the two genes would alleviate the tension that creates the supercoiling. Another one of our methods was to use a dCas9 clamp between the RFP and GFP to clamp down between the two genes and stop the supercoiling.
In order to see whether or not the problem of supercoiling was reduced, we had to first construct the plasmid circuits used for our experiment by using Golden Gate Assembly. First, we designed primers to amplify our parts off current iGEM parts using the online DNA design editor, Benchling.For our experiment, we have used RFP and GFP and parts from the iGEM inventory: BBa_J04450 and BBa_I13522, respectively. These parts were amplified off and ligated together in varying orientations (see picture designs) with a spacer part between them (either 500bp spacer, 1000bp spacer, or a dcas9 clamp site spacer) and finally a vector, or backbone piece, with Chloramphenicol or Kanamycin resistance.
The RFP gene and the GFP gene with transcription occurring in opposite directions away from the dCas 9 binding sites, which separate the two genes.
The GFP gene being transcribed towards the dCas9 binding sites and the RFP gene, while the RFP gene is being transcribed away from the GFP gene.
The RFP gene and GFP gene while transcription is occurring towards each other, as well as in the direction of the dCas 9 binding sites.
The RFP gene being transcribed towards the dCas 9 binding sites and the GFP gene, while the GFP gene is being transcribed away from RFP gene.
The RFP gene and GFP gene are switched. The RFP and the GFP with transcription occurring in opposite directions away from the dCas 9 binding sites, which separate the two genes.
RFP and GFP are switched. The RFP gene being transcribed towards the dCas9 binding sites and the GFP gene while the GFP gene being transcribed away from the RFP gene.
RFP and GFP are switched. The RFP gene and GFP gene while transcription is occurring towards each other, as well as in the direction of the dCas 9 binding sites.
RFP and GFP are switched. The GFP gene being transcribed towards the dCas 9 binding sites and the RFP gene, while the RFP gene is being transcribed away from the GFP gene.
