Part:BBa_K2946010

TS1

Part one of a logic AND gate circuit based on trans - splicing events (TS). This reaction is designed to take place between the transcripts of two exogenously introduced modules. Expression of module TS1 is driven by the SSX1p promoter, which was used by Nissim et al. and is active in HEK293 cells (1). Our circuits are based on TS events designed to take place between the transcripts of two exogenously introduced modules, thus allowing full degree of freedom in selecting the optimal guiding sequences. Here, downstream to exon 1 of mKate2 we have introduced the first 198 bp of the miR1 intron employed by Nissim et al. (1), including the 5’ basal stem to serve as the TS guiding sequence to module TS2. This sequence is followed by the 34 bp linker of the first module in (1) and the HSV1pA poly A site. This pre- mRNA is designed to attach to another synthetic pre-mRNA (module TS2 BBa K2946011) if present, in order to create a full mRNA of our gene of interest (GOI - MKate2) through trans-splicing. By itself it will not form a transcriptable RNA code and will not produce protein.

Design and Construction of Circuits

In order to achieve our goal, we first designed and constructed the Trans Splicing (TS)-based AND gate circuits, LoGENEgate. We engineered two genetic modules such that each was regulated by a separate promoter. Module 1(Figure 1) consists of P1 (SSX1p), which regulates the expression of exon 1 of mKate2 as well half of the intron that include a strong donor splice site and linker.

In this system, the output protein is expressed only when the promoters regulating both modules are mutually active. However, when only one of the promoters is active or when none of the promoters are active, they cannot produce any functional protein. thus, standing alone, is permanently in state ‘0’. We defined two different states for this circuit: in state [1,0], module 1 is active, while module 2 is inactive (off); and in state [1,1], both module 1 and module 2 are active (on).

Results

Expression in HEK293T cells - the data below display the transient transfection of HEK293T cells with plasmids containing LoGENEgate by flow cytometry. Any expression below the negative control (<3.09%) is irrelevant, hence amounts to 0% expression of GOI. As expected in [1,0] states there is no product, 0% expression