Team:Dundee Schools/Proof
Dundee Schools
Proof of Concept
S.O.R.D.
We designed S.O.R.D. as an RNA secretion device. It is comprised of osmY (protein naturally secreted by E. coli) and Hfq (RNA binding protein) fused together. This device is what will transport our spiRNA out of our E. coli and to the target bacteria.
In the labs we wanted to test if S.O.R.D. could be secreted, so we used a western blot to do so.
Western blotting indicated that our S.O.R.D. was being secreted entirely. This meant for future plans that S.O.R.D. should theoretically secrete and be capable of carrying RNA on its journey out of the cell.
spiRNA
Our RNA silencing agent spiRNA is what will attach to a specific gene on an mRNA strand in the target cell. We decided to test spiRNA by targeting the fliC gene which forms the FliC protein. This is the repeating subunit of the flagellum. By targeting this gene the bacterium’s motility should be greatly decreased and therefore easy to see if our device works. We did this by expressing a spiRNA containing a fliC targeting sequence inside a target bacterium.
Result:
Our results showed that the bacteria became less motile as there wasn’t as much expansion on the plate as the control experiment. This is strong indication that spiRNA effectively targeted fliC mRNA and reduced its translation, proving the principle of it being able to target specific genes with downstream functional effects. This means that by changing the targeting sequence, in theory we could target any gene at will.
