RNA Background RNA has traditionally been considered as mainly a carrier of information between the gene and the final protein product. However, recent evidence is accumulating for alternate functions for RNA—namely, RNA molecules as regulatory elements in the cell. An example of this is RNA that regulates gene expression in the cell such as ribozymes, siRNA, and riboswitches.
Riboregulators New insights into this regulatory role of RNA have allowed for engineering new RNA molecules which control cell behavior through base-pairing or molecular interactions to other RNA molecules or appropriate ligands, respectively. One such engineering approach which enables post-transcriptional control of gene expression is the riboregulator, which was initially developed by Collins and coworkers. The riboregulator is composed of two interacting parts—the cis-repressive sequence and the trans-activating sequence. The cis sequence is directly upstream of the ribosome binding site (RBS) of the gene under the control of the riboregulator and is complimentary to the RBS. This sequence causes a stem-loop structure to form at the 5’-untranslated region of the mRNA which prevents ribosome binding and thus translation. The trans RNA is complementary to the cis sequence, and when bound to this sequence, the RBS site becomes exposed, allowing for ribosome binding and translation.
Riboregulator Integration in Our Phage/E.coli System The riboregulator is the decisive factor in our system which determines what happens to the cell once it is infected by phage. We aimed to design three types of riboregulated systems (one for each protein N, Q, and Cro). For instance, for the N protein-regulated system, the phage genome would have a mutant version of the N gene as well as an N gene suppressed with a cis-repressive sequence would be additionally integrated into the genome. When the phage infects a wild-type E.coli cell, nothing should happen. However, when it infects a cell with the trans-activating non-coding RNA, the cell should lyse because the RBS of the N gene is no longer sequestered by the cis-repressed RNA loop.
Experiments with Riboregulators In order to have a working system in which phage infection outcome is controlled, the expression of genes under the control of a riboregulator must be determined. Once the amount of N, Q, or Cro necessary to switch the system from lysis to lysogeny (or nothing) is determined, an appropriate riboregulator can be integrated into the system which allows for the correct amount of protein to be made in the trans-activating cells.
Before combining the phage protein expression with the riboregulator part of the system, it is necessary to engineer various riboregulators and determine the percent of protein (green fluorescent protein or GFP) in the presence and absence of trans-RNA. By quantifying the riboregulated GFP system, we could then apply the results to each protein.
- The team completed digestion of biobrick parts P0440 (tetR gene and terminator); J23100 (strong constitutive promoter); J23116 (weak constitutive promoter); B0015 (terminator); and P1010 (2K3 plasmid backbone).
- We also assembled the first base plasmid with B0015, J23116, and P0040 in the 2K3 plasmid (replacing the ccdB gene) via ligation.
- The design of 8 total cis-repressive regions for the riboregulator have been complete. The designs were based on previously-successful riboregulators from the Collins group as well as the 2006 UC Berkeley iGEM team.
- The GFP gene was successfully extracted from E0040, to be used for inserting before the B0015 terminator in the 2K3 plasmid constructed last week.