Team:BostonU/Design
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. We utilized the recombinase system BLADE. BLADE allows for digital activation of different genes by using inducible recombinase proteins to excise DNA. When different combinations of recombinases are activated, different combinations of DNA are excised. Based on what parts of the circuit are excised decides which of several gRNA's to release. Once a gRNA is released, it will 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 gene. The results of BLADE can be seen in the graph below.
As seen above, this is very similar to our system. Except instead of activating different genes, we want to integrate the same gene to different levels based on the signal input. We adapted BLADE by integrating our well characterized parts into its framework. Instead of each gRNA in the circuit corresponding to the operator upstream of a different gene, each unique gRNA would all correspond to one gene. The difference is that each copy of the gene has a different number of target sequences for the activator to bind to, and each combination corresponds to a different one of the unique gRNA's released by BLADE. gRNA 1 corresponded to a reporter with one target sequence, and gRNA 2 corresponded to the same gene but with two of the target sequence. Releasing gRNA 1 turned the gene on to a small degree, and gRNA two turned it on to a large degree. BLADE allows us to integrate more signals and more recognition site, achieving a full two signal truth table. Since the recombinase circuits that release the different gRNA is completely digital, (the recombinases are activated by the digital prescience or absence of a signal such as a hormone and once activated, are extremely efficient) the system was a merger of digital signals giving rise to different levels of analog gene expression, as stated in our goal. A diagram of these circuits can be found below.
