Part:BBa_K4761101

pET expression vector

pET plasmid vector. Expression vector for rhodioloside production and RNA Thermometer. It has medium copy number and Amp resistance. Note that it has no T7 promoters. BamHI and KpnI are necessary for gene installation.

pET vector system is a powerful and widely used system for expressing recombinant proteins in E. coli. For this vector used in 2023 HiZJU-China, it shares characteristics with another vector pZE instead.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2
  Illegal XhoI site found at 85
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 402
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 987
  Illegal SapI.rc site found at 2647
  

Characterization

In 2023 HiZJU-China, this vector is originally used as a carrier for UGT85A1(BBa_K4761002), which encodes a speed-limiting enzyme in the rhodioloside production pathway.

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

Fermentation has proved its expression capability by elevating the rhodioloside yield from 0 to 7mg/L. See Results in 2023 HiZJU-China.

This vector is also used in RNA Thermometer module. As the absence of T7 promoters, it has flourescence hard to observe by naked eye but detectable by microplate readers.

Fig.2 RNA Thermometer results based on pET vector.