Team:BostonU/Design
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:
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, we integrated our characterized analog parts into recombinase based circuit. These circuits use inducible recombinase proteins to excise DNA marked by specific sequences. When different combinations of recombinases are activated by digital signals, different combinations of the gene circuit are excised. Based on what parts are excised decides which one of several gRNA's to express, guiding the dCAS9-VPR activator to its corresponding operator. When a new combination of recombinases is 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 could have a second operator multimerized twice. Thus when gRNA 1 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 can increase the number of inputs and outputs. The finished product relied on digital singnals, (the recombinases are activated by digital signals such as a hormone prescense) and gave rise to different levels of analog gene expression, as stated in our goal.
