Team:BostonU/Modeling/Split Sites

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Matlab Code

Split Site Identification

Split Site Identification

Our team had three main criteria for making split site decisions. We decided that the important things to avoid were the inner core of the protein, secondary structures, and catalytic residues. We hypothesized that splitting in these areas may disrupt the function of the protein even when the protein halves were dimerized. In order to determine where these regions were on the integrases, RDFs, and saCas9, we used multiple resources. Firstly, we utilized a model developed by a graduate student in our lab to predict the hydrophobicity of a chain of amino acids. Since the inner core of the protein is typically very hydrophobic, we were able to use this model to ensure that we only chose split sites that were on the hydrophilic surface of the protein. Next, we used an online tool called JPRED. This resource uses the primary structure of a protein to predict the locations of alpha helices and beta sheets, the secondary structure. Using JPRED, we chose sites that were not in any secondary structures of the proteins. Lastly, we wanted to avoid catalytic residues so we looked through literature and were sure to avoid sites that corresponded to important catalytic regions of the protein. All in all, we decided on 8 split sites for each integrase and for saCas9 and 4 split sites for the RDFs. We wanted to test each split site with each of the three dimerizable domains and in each orientation. For instance, if TP901-1 was split at AA253, we wanted to try fusing FKBP to AA1-253 and FRB to AA254-486 as well as FRB to AA1-253 and FKBP to AA254-486.

Dimerizable Domains

We decided to try three different dimerizable domains with our protein splitting procedure. FKBP/FRB induced with rapalog, PYL/ABI induced with abscisic acid, and CRY2/CIBN induced with blue light. All of these systems have been studied in literature in the past, and have their own benefits. The CRY2/CIBN system is incredibly fast, the domains can come together in as little as 300 microseconds. Additionally, blue light has a very high resolution and can be delivered to cells with higher accuracy than chemically induced systems. However, these domains are the largest out of the three, which may inhibit efficient binding when fused to smaller proteins such as RDFs. FKBP/FRB is the most well documented dimerizable system and has been previously shown to be effective. We are using a slightly altered inducer of the standard rapamycin called rapalog in order to decrease non-specific binding. The domains bind the tightest of the three and also have the smallest domains. PYL/ABA is a dimerizable system found naturally in plants and is completely orthogonal to mammalian systems. Once bound in the presence of the inducer, abscisic acid, the domains can be split by washing the system and waiting 24 hours. All of these systems are orthogonal, an important aspect for future research into genetic logic circuits with the integrase RDF system.