Part:BBa_K3504009

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

Characterization

Figure 1.in vitro replicon enhancement method: Using transfected Jurkat cells with replicon RNA which was encoded for mCherry and grown in cell culture under the SGP. Then during serial passage as shown in the flow cytometry histograms, the top 20 percent of mCherry were sorted around every 10 days. this has resulted that cells expressing higher levels of repoter gene were enriched. finally the 5th sort cells were seperated from the rest for replicon sequencing.

Figure 2.Mutation identification:Positive mCherry cells was reverse transcribed to cDNA,after that Nsp1 to 4 and the SGP were magnified by seven pairs of primers and amplicons and later on engineered into DNA of the plasmid and changed into E.coli to be amplified. The lower left part schematic illustrates roughly the locations of point mutations in the 5th sort in NSP2 & NSP3.

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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2760
  Illegal SpeI site found at 2708
  Illegal PstI site found at 5089
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2760
  Illegal NheI site found at 5960
  Illegal NheI site found at 7290
  Illegal NheI site found at 8107
  Illegal SpeI site found at 2708
  Illegal PstI site found at 5089
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2760
  Illegal BglII site found at 2882
  Illegal BamHI site found at 2744
  Illegal BamHI site found at 3107
  Illegal XhoI site found at 6124
  Illegal XhoI site found at 6185
  Illegal XhoI site found at 6226
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2760
  Illegal SpeI site found at 2708
  Illegal PstI site found at 5089
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2760
  Illegal SpeI site found at 2708
  Illegal PstI site found at 5089
  Illegal NgoMIV site found at 3549
  Illegal AgeI site found at 5665
  Illegal AgeI site found at 6034
  Illegal AgeI site found at 6361
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1635
  Illegal BsaI site found at 6223
  Illegal BsaI site found at 6241
  Illegal BsaI.rc site found at 3012
  Illegal BsaI.rc site found at 3659
  Illegal BsaI.rc site found at 5750