We want to optimize linker length for both the linker between the CD79A/B and Gal4VP16 (which we call the TCS linker) and the linker between Syk and TEVp (which we call the Syk linker). We also want to optimize the position of the Tev Cleavage Site (TCS) within the TCS linker. To do that, we first optimize the TCS linker. We then optimize the Syk linker.
TCS Linker Optimization:
Purpose: To design a series of flexible linkers (each containing a TEV cleavage site) for use between CD79A/B and Gal4VP16 that covers a large portion of the search space. The goal of testing various linkers is to find one with an optimal on/off ratio when used in the BCR/Tango system.
Search space:
Left designates addition to 5' end and right designates 3' addition.
Rationale for linker choices:
Linker amino acid sequences for 9aa and 15aa linkers were taken directly from "Optimizing the stability of single-chain proteins by linker length and composition mutagenesis" (Clifford R. Robinson and Robert T. Sauer, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC34497/). Linker amino acid sequences for 3aa, 6aa, and 12aa linkers were based on the linker patterns presented in the paper above while taking into account the need for consistent start and end sequences (Q sites). Q8 was chosen as the 5' Q site and Q6 was chosen as the 3' Q site. All linkers are glycine-rich, flexible linkers.
TEV cleavage site was selected from the original Tango system paper ("The genetic design of signaling cascades to record receptor activation", Barnea et al., http://www.pnas.org/content/105/1/64.full). TCS(L) was selected because it had the greatest on/off ratio of the tested TCS variants.
Linker design:
DNA oligomers were designed for easy insertion between CD79A/B and Gal4VP16 (from construct CD79A/B-pTET:RFP-Gal4VP16) using a Golden Gate reaction. Thus, linkers were designed according to the following pattern:
Spacer - BsaI - Extra nt - Left linker - TCS - Right linker - Extra nt - BsaI - Spacer
The 5' end of each linker used a Q8 overhang and the 3' end a modified Q6 overhang that included the same base pairs but was inserted in the opposite direction. Based on a detailed Golden Gate protocol ("Combinatorial DNA assembly using Golden Gate cloning", Engler C and Marillonnet S, http://www.ncbi.nlm.nih.gov/pubmed/23996445), directionality of the Q site should not significantly affect the success of the Golden Gate reaction.
Feb 22 - DNA constructs for linkers designed in silico: compatible with cloning into CD79A/B-mRFP-Gal4VP16 construct to be cloned via PCR.
Syk Linker Optimization: