Difference between revisions of "Part:BBa K3504009"

Revision as of 08:46, 13 October 2020

Alphavirus replicon NSPs- Equine Encephalitis virus

Part Description

A composite of parts (BBa_K3504000,BBa_K3504001,BBa_K3504002,BBa_K3504003) and CMV promoter which form ,as a unit, the main constituent of alphavirus replicon which as a whole can give the circuit self-replicating ability

Usage

Characterization

Figure 1.Method for in vitro replicon evolution: Jurkat cells were transfected with replicon RNA encoding mCherry under the SGP and grown in cell culture. The top 20% of the mCherry+ population were sorted for approximately every 10 days during serial passaging as indicated by the flow cytometry histograms, leading to an enrichment in cells expressing high levels of the reporter gene. Cells from the 5th sort were isolated for replicon sequencing.

Figure 2.Identification of mutations: Total RNA from mCherry positive cells was extracted and reverse transcribed to cDNA. Then, nsP1–4 and the subgenomic promoter were amplified by seven pairs of specific primers and amplicons from Loci 1–7 were engineered into plasmid DNA and transformed into E. coli for amplication. Six clones from each locus were randomly picked for Sanger Sequencing. Schematic at bottom left shows the approximate locations in nsP2 and nsP3 where point mutations were identified in 5 mutant alleles with c2 harboring two linked mutations.

Improvements

Using information in literature we were able to increase the replicon cloning and functional ability by adding G110G, G763R Mutation to NSP2 and adding K94E, S243G,E255D,V305M Mutation to NSP3

Figure 1. Nsp2 G110G Amino acid mutation.

Figure 2. Nsp2 G763R Amino acid mutation.

Figure 3. Nsp3 K94E Amino acid mutation.

Figure 4. Nsp3 S243G Amino acid mutation.

Figure 5. Nsp3 E255D Amino acid mutation.

Figure 6. Nsp3 V305M Amino acid mutation.

References

Li, Y., Teague, B., Zhang, Y., Su, Z., Porter, E., Dobosh, B., ... & Weiss, R. (2019). In vitro evolution of enhanced RNA replicons for immunotherapy. Scientific reports, 9(1), 1-10. Sequence and Features