Part:BBa_K2933177

Tac promoter+RBS a+Linker g+GST+Linker e+ARL-1

This part consists of Tac promoter,RBS and protein coding sequence (GST+Linker e+ARL-1),and the biological module can be built into E.coli for protein expression.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1221
  Illegal PstI site found at 1335
  Illegal PstI site found at 1386
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1221
  Illegal PstI site found at 1335
  Illegal PstI site found at 1386
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1221
  Illegal PstI site found at 1335
  Illegal PstI site found at 1386
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1221
  Illegal PstI site found at 1335
  Illegal PstI site found at 1386
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 181
  

Usage and Biology

This composite part is made up with six basic parts(Tac promoter，RBS a , Linker g, GST, Linker e and ARL-1). It encodes a protein which is ARL-1 fused with GST tag. The fusion protein is about 57.0 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of ARL-1 and combine the two parts with the cutting site of Prescission Protease. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pGEX-6p-1 and the vector to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

Figure 1. Left: The PCR result of ARL-1. Right: The verification results by enzyme digestion.