Part:BBa_K4451021

MutaT7 Test Cassette ACG

BBa_K4451021 (MutaT7 ACG Test Cassette) is a composite part containing streptomycin and chloramphenicol resistance genes, which has been designed to function as a reporter of the in vivo activity of the cytosine deaminase-T7 RNA polymerase fusion protein, BBa_K4451003. This construct was heavily inspired by the similar cassette used by Moore, Papa, and Shoulders (2019) to demonstrate function of their ‘MutaT7’ continuous in vivo directed evolution system, which formed the basis of iGEM Sheffield 2022’s project.

By default, both antibiotic resistance genes are not translated due to a non-functional ‘ACG’ start codon preceding their respective coding sequences. When BBa_K4451003 is active in the cell, the T7 RNA polymerase subunit begins transcription at the T7 promoters flanking SmR. Localised cytosine deaminase activity is expected to enable the false start codon ACG to be mutated to ATG (via an AUG intermediate), thereby conferring streptomycin resistance to the cell at a higher frequency compared to the background rate of mutation.

In order to confine the region of hypermutation to the gene of interest (in this case, SmR), the synthetic termination signal, Tz (Mairhofer et al, 2015), is used here to insulate the rest of the construct from deaminase activity. The upstream CmR gene is also not translated unless the false start codon is mutated to ATG, and therefore rescue of chloramphenicol resistance at a higher frequency than a deaminase-free control would indicate transcriptional read through of the Tz terminator.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1599
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal PstI site found at 1599
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 850
  Illegal XhoI site found at 1731
  Illegal XhoI site found at 1937
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1599
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1599
  Illegal AgeI site found at 561
  
• 1000
  COMPATIBLE WITH RFC[1000]