Project Specific Changes

As part of the project we looked to spread synthetic biology throughout the community. We approached this feat through several different avenues.

Our project came into fruition after extensive debate and discussion, the idea of introducing a mechanism by which RNA can be propagated is both conceptually and theoretically appealing. Using our replicon system to target production of a siRNA directed against a target (DPP-IV) shows just one of many applications that our system holds. Initially, it was not so straight forward when considering additional elements that we might require – for safety of the system was not on our minds. However, after due deliberation we decided to embark and ask questions and most importantly, seek answers. We started small and simple, with the obvious question on how people perceive our system? We decided to conduct a Q + A session, in which academics, students and the wider public were in attendance. Whilst lost of interesting questions were asked, we collectively became stuck when faced with the following question:

What have you done to ensure your system is safe?

Having reflected on this question, we realized that we failed to consider how our system would be safe - as one of the main applications of our system was to help treat Diabetes, we had to re-think and go back to the drawing board. We came up with two ideas which we feel will greatly improve the safety, but also the efficiency of our system

Aptazyme

We decided to use a theophylline induced aptazyme, this essentially acts as a kill-switch if our system becomes unstable - it is possible to cleave our siRNA transcript and prevent our system from functioning. A general descriptor for the Aptazyme is below:

An aptazyme is an RNA based switch that operates by ribozyme-mediated cleavage of RNA. This part derives from Hartig et al., 2002 and requires the addition of theophylline to induce hammerhead ribozyme activation and cleavage, as depicted in Figure 1a. The part sequence is modified to contain a stop codon at the end, as an RBS is present.

In addition, we introduced another safety device, essentially this would rate limit the level of RNA dependent RNA polymerase (RdRp) production. Our design of RNA dependent RNA polymerase essentially incorporated an MS2 bacteriophage coat protein, linked via a P2A linker protein. A general descriptor for the part we used is below: MS2 bacteriophage coat protein part derives from Fussenegger et al., 2012. The MS2 coat protein binds a specific stem-loop structure, referred to as the MS2 box. This acts as a silencing mechanism of RNA through translational repression (Ni et al., 1995). The full sequence found in Fussenegger et al., 2012 has been retained, as previous analysis has indicated alteration of MS2 coat protein reduces cooperative protein-RNA binding (Uhlenbeck et al., 1995). MS2 is also frequently used in biochemical purification of RNA-protein complexes and is combined with GFP to detect RNA in living cells.