Difference between revisions of "Team:BostonU/Design"

In order to make genes activate in response to certain signals, our system first needed a method to activate genes in general. We chose CRISPR/dCAS9-VPR as an activator. We chose dCAS9 due to its ease of use and its ability to target specific DNA sequences. dCAS9-VPR targets specific sequences by binding to specialized RNA. Part of this RNA (gRNA) contains 20 base pairs that will act as a guide, guiding the dCAS9 to the complimentary 20 base pairs found upstream of a gene one wishes target. This can be seen in the info-graphic below:

Once we were able to activate genes, we then expanded our system to activate genes to different levels, thereby achieving the graded analog expression level that we desired from our system. To accomplish this, we multermerized the 20 base pair target sequence, placing multiple copies of the target sequence upstream of the gene. By varying the number of copies, we were able to create a gradient of expression. The more target sequences we added, the more the gene was activated. This is illustrated in the image below:

Finally, once we completed phase one and two, we expanded our system once again, integrating our characterized parts into recombinase based circuit. These circuits digitally control transcription. Inducible recombinase proteins can be activated by digital signals, and once active, excise DNA marked by specific sequences. When different combinations of recombinases are activated by digital signals, different combinations of a gene circuit are excised. Based on what parts are excised decides which one of several gRNA's to expressed. The chosen gRNA bind to a dCAS9-VPR and guide the activator to the gene with the corresponding operator. When a new combination of recombinases are activated, a different gRNA is released, guiding the activator to a different operator. This circuit is co-transfected with plasmids containing the same gene of interests, but different operators. The first plasmid could have one operator corresponding to the first gRNA; the second plasmid have a different operator multimerized twice. Thus when gRNA one is expressed, we see expression of the gene of interest, and when gRNA two is expressed, we see an increase in expression. A diagram of this process can be found below. As we integrate more signals and more recognition site, we could increase the number of outputs and inputs. The finished product relied on digital activation, (the recombinases are activated by the digital prescience or absence of a signal such as a hormone) and gave rise to different levels of analog gene expression, as stated in our goal.