Part:BBa_K2946015

TS11 + TS12

By exploiting the phenomenon of mRNA trans-splicing (TS) the bimolecular joining of exons from different mRNA species (our two sets of basic parts) will create a full functioning Gene of interest (GOI) only when they attached.(3). TS can be targeted to pre-selected transcripts (referred to as ‘targeted TS’) by promoting base pairing between corresponding introns flanking the exons to be trans-spliced. In the composite part we are presenting, we created a logic ‘AND’ gate two-module system. An ‘AND’ gate will generate an output only when all the inputs, module 1 and 2, are active. Module 1, driven by a synthetic promoter P1, encodes the 5’ end of the GOI (Mkate2 in this case), including the translation initiation codon, referred to as exon 1, followed by the TS ‘target’ intron (Figure 1).

Critically, no mature, potentially hazardous protein can be translated from this module, ensuring the desired ‘0’ state. Module 2, governed by promoter P2, encompasses the TS ‘guide’ intron, followed by exon 2, which encodes the rest of the protein. Similarly, module 2 cannot produce any functional protein and, standing alone, is permanently in state ‘0’ too. Module 2 can possess tandem repeats of the TS guide sequences to increase the prospects for productive TS events. Using this design, the system becomes fully dependent on the synchronized function of both promoters P1 and P2: the risk of translation of the intact TF or the polypeptide product of any GOI in cells in which promoter P1 only or promoter P2 only is active is utterly obviated. A major technological challenge facing the ‘conventional’ use of TS, when the target sequence in the target cell is the transcript of the endogenous gene to be corrected, is how to successfully compete with cis-splicing (2). Applying TS as we have done, when both transcripts are encoded by exogenous genes, unintended cis-splicing can be entirely avoided. This can be achieved by depriving the respective target genes of the intronic elements that are mandatory for completing an efficient splicing process: the acceptor splice site, the poly-pyrimidine tract and the branch point for module 1 and a donor splice site for module 2. This pre- mRNA designed to attach to another synthetic pre-mRNA (module TS12) 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

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