Part:BBa_K5048066

LAP-GST

Codes for the membrane protein of L.innocua, which can bind to Hsp60 on the membrane of small intestinal epithelial cells, and another type of E.coli that we have engineered. Codon optimized for expression in E.coli, and a GST tag was added to improve the solubility and tag specificity of the protein.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1429
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1429
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2472
  Illegal BglII site found at 2570
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1429
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1429
  Illegal NgoMIV site found at 678
  Illegal NgoMIV site found at 1944
  Illegal AgeI site found at 310
  Illegal AgeI site found at 1966
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 2292
  

Design

GST: Glutathione S-transferase from Schistosoma japonicum. We implement the GST tag for better specificity for protein detection. We also try to introduce LAP-GST fragment into plasmid backbone pET-28a and pBAD(Fig 1)(Fig 2).

Fig 1 Map for plasmid LAP-GST

Fig 2 Map for plasmid pBAD-LAP-GST

Source

We used amplified segments from vector pBAD and plasmid LAP-His(BBa_K5048058).

Experiments

This part acts as an adhesin that can adhere to HSP60.

The overall experiments we conducted on BBa_K5048066 include:

1. Verification of PCR amplification products.

Result

Verification of PCR product

We amplified segments GST and pET-LAP backbone(not succeeded yet) for the LAP-GST segment(Fig 3).

Fig 3 The segments that amplified successfully for genetic circuits
A. The agarose gel electrophoresis of GST for plasmid LAP-GST and LAP segment for plasmid pBAD-LAP. GST: 689bp; LAP: 2652bp.

Applications of BBa_K5048066

This part acts as an adhesin that can adhere to HSP60.