Qbeta Rep

I also call this ‘in vivo spiegelman’s monster’. The mission is to achieve orthogonal RNA replication with a high mutation rate using Qbeta replicase.

Qbeta Map

The beauty of the Qbeta phage (above)

An in vivo spiegelman monster has several advantages to current technologies. First, qbeta is *nearly* completely orthogonal from cellular RNAs. This built in orthogonality means that selection at a high mutation rate can be achieved in vivo WITHOUT complex polymerase building and clever genetic systems. Because of that, qbeta can also be used in a PACE system with less cells (qbeta creates more progeny than M13), continuously without continuous cells (PACE depends upon this to avoid host mutation , which is not a problem with the orthogonality of qbeta) and with a higher mutation rate (qbeta mutation rate is 1.4 × 10^−4 and the E coli host is about 2.2 × 10^-10 per nucleotide. PACE system only increases mutation rate 100 fold).

Overall, if I can show that qbeta RNA can be replicated in a cell for some number of generations by it’s own polymerase, I can begin developing orthogonal replication systems and PACE systems for accelerated evolution.

To do this, I am deleting part of the maturation gene, which codes for the entry protein and lysis protein, then doing ‘burst expression’ with araBAD. If I can still detect RNA after a few generations, and cannot detect RNA in the negative control, this will be enough for a proof-of-concept.

I have recieved qbeta phage from attostar and will be doing RT-PCRs to create aforementioned genetic construct as soon as I can afford some reverse transcriptase.


Qbeta-RNA_syn Map

A hypothetical RNA replication cassette with ZeoR as a selection marker (chosen for small size, above)


qbeta-zeo_syn

qbeta-syn with ZeoR gene (above)


qbeta_syn

qbeta-syn without ZeoR gene (above)