Part:BBa_K2946011

TS2

Part two of a logic AND gate circuit based on trans - splicing events (TS). This reaction designed to take place between the transcripts of two exogenously introduced modules. This module is designed to facilitate optimal targeted TS with the transcript of module TS1 to generate the intact mKate2 coding sequence. Its expression is driven by the H2A1p promoter (1), followed by a 40 bp linker (positions 421-460 in the kanamycin resistance gene of thermophilic bacillus from plasmid pTB913 (GenBank accession K02551.1) and the remaining 219 bp of the miR1 intron, including the 3’ basal stem as the TS1 TS binding domain (BD) and the full acceptor splice site, followed by mKate2 exon 2, 40 bp linker (positions 361-400 in the kanamycin resistance gene), BamHI and HindIII restriction sites and the HSV1pA poly A site.This pre- mRNA designed to attach to another synthetic pre-mRNA (module TS1) if present in order to create a full mRNA of our gene of interest (GOI - MKate2) by 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. Module 2 consists of P2 (H2A1p), which regulates a TS guiding sequence, the acceptor splice site and mKate2 exon 2(Figure 1)

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