Part:BBa_K4761102

pCS expression vector

pCS plasmid vector. Expression vector for rhodioloside production. It has Kan resistance and medium copy number. BamHI and KpnI are necessary for gene installation.

The pCS vector system is an efficient system for expressing recombinant proteins in E. coli at low temperatures, typically 15°C. It is based on the E. coli cold-shock gene cspA, and also includes part of the lac operon regulatory system.

However, in rhodioloside production in 2023 HiZJU-China, it is also seen to successfully express pgm-galU(BBa_K4761201) and UGT85A1(BBa_K4761002) under 37℃.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2157
  Illegal XbaI site found at 864
  Illegal SpeI site found at 934
  Illegal PstI site found at 1683
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2157
  Illegal SpeI site found at 934
  Illegal PstI site found at 1683
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2157
  Illegal BglII site found at 1896
  Illegal BamHI site found at 2
  Illegal XhoI site found at 2077
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2157
  Illegal XbaI site found at 864
  Illegal SpeI site found at 934
  Illegal PstI site found at 1683
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2157
  Illegal XbaI site found at 864
  Illegal SpeI site found at 934
  Illegal PstI site found at 1683
  Illegal NgoMIV site found at 1232
  Illegal AgeI site found at 432
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

In 2023 HiZJU-China, pCS vector is originally used for expression of pgm-galU(BBa_K4761201). pgm-galU is the UDP-glucose generation unit, enhancing the rhodioloside production.

Fig.1 Verification for original backbones and genes. pCS backbone marked out.

Fermentation experiment shows that pgm-galU expressed by pCS boosts the rhodioloside yield(7 to 12mg/L on 36h). And statistics show that when UGT85A1 is installed on it, it showed remarkable expression results, proving its expression activity.

Fig.2 HPLC analysis result for fermentation product on 36h. (a)pSA-ARO10-adh6+pET-UGT85A1; (b) +pCS-pgm-galU based on (a). Notably clear increase of the peak height; (c) pCS-UGT85A1, this peak is for another byproduct due to excessive UGT expression.