Part:BBa_K4761103

pACYCDuet dual expression vector

pACYCDuet dual expression vector for rhodioloside production. It has high copy number and Kan resistance. It is a bacterial vector with a p15A origin for the co-expression of two genes.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 112
  Illegal XbaI site found at 2749
  Illegal PstI site found at 131
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 112
  Illegal NheI site found at 1908
  Illegal PstI site found at 131
  Illegal NotI site found at 149
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 112
  Illegal BglII site found at 305
  Illegal BamHI site found at 106
  Illegal XhoI site found at 354
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 112
  Illegal XbaI site found at 2749
  Illegal PstI site found at 131
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 112
  Illegal XbaI site found at 2749
  Illegal PstI site found at 131
  Illegal NgoMIV site found at 324
  Illegal AgeI site found at 566
  Illegal AgeI site found at 1994
  Illegal AgeI site found at 2317
  
• 1000
  COMPATIBLE WITH RFC[1000]

Results

pACYCDuet is used for Copy Number Optimization module in 2023 HiZJU-China. The functional parts are linked on the vector using Gibson Assembly. The backbone could be obtained using primers BBa_K4761513 and BBa_K4761514(installing ARO10-adh6) or BBa_K4761518 and BBa_K4761519(installing UGT85A1).

Fig 1 Verification of backbones and genes for Gibson Assembly. pACYCDuet backbone on Lanes 7 & 8.

This plasmid is able to be transformed into BL21(DE3) strain and express with efficiency. For details, see Copy Number Optimization results page of 2023 HiZJU-China.

Fig 2 Characterization for successfully constructed plasmid pACYCDuet-ARO10-adh6-UGT85A1.

By installing BBa_K4761200 and BBa_K4761002 on this vector, it is observed to have higher salidroside yield than original fermentation strain pSA-ARO10-adh6+pET-UGT85A1.

Fig 3 HPLC results for (a)pSA-ARO10+pET-UGT85A1 and (b)pACYCDuet-ARO10-adh6-UGT85A1. Salidroside peak hard to observe due to byproducts from excessive UGT85A1 expression.