Part:BBa_K4451019

CD + AD MutaT7 Cassette

This part is a composite of two base deaminase-T7 RNA polymerase fusions placed in series, each under the control of a pJKR-L-TetR-derived aTc-inducible promoter. This part represents the hypermutation plasmid designed by Sheffield 2022 as part of the ‘rEvolver’ three-plasmid modular directed evolution system, inspired by the MutaT7 system developed by Moore et al. (2018).

BBa_K4451003 encodes a cytosine deaminase-T7 RNA polymerase chimeric protein. EvoAPOBEC1-BE4max, a variant of the cytosine deaminase APOBEC1, was developed through phage-assisted continuous evolution by Thuronyi et al. (2019). BBa_K4451004 encodes an adenosine deaminase-T7 RNA polymerase chimeric protein. TadA* variants were recently developed through directed evolution by Gaudelli et al. (2020) to exhibit higher editing efficiency (in the context of sgRNA-directed base edititing). Both base deaminases have been tethered to the phage-derived T7 RNA polymerase via a (G3S)7 link.

Both evoAPOBEC1-BE4max and TadA* have previously been used in the context of T7RNAP fusions by Evry Paris-Saclay 2021 as part of their Evolution.T7 system (BBa_K3766008 and BBa_K3766004, respectively). The base deaminase fusions used in rEvolver differ slightly in that all CDS within this cassette have been codon-optimised for expression in Vibrio natriegens, a marine bacterium that is capable of shorter doubling times than E. coli. Sheffield 2022 showed that due to the faster doubling time of V. natriegens, one would expect fixation of beneficial alleles to occur at a faster rate than E. coli (https://2022.igem.wiki/sheffield/model).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 74
  Illegal EcoRI site found at 3834
  Illegal PstI site found at 768
  Illegal PstI site found at 4369
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 74
  Illegal EcoRI site found at 3834
  Illegal NheI site found at 2777
  Illegal NheI site found at 2825
  Illegal NheI site found at 3737
  Illegal NheI site found at 6318
  Illegal NheI site found at 6366
  Illegal PstI site found at 768
  Illegal PstI site found at 4369
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 74
  Illegal EcoRI site found at 3834
  Illegal XhoI site found at 3755
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 74
  Illegal EcoRI site found at 3834
  Illegal PstI site found at 768
  Illegal PstI site found at 4369
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 74
  Illegal EcoRI site found at 3834
  Illegal PstI site found at 768
  Illegal PstI site found at 4369
  Illegal NgoMIV site found at 1617
  Illegal NgoMIV site found at 5158
  Illegal AgeI site found at 123
  Illegal AgeI site found at 816
  
• 1000
  COMPATIBLE WITH RFC[1000]