Part:BBa_K3588020

Synthetic Adhesin

This part is a synthetic adhesin (SA) expressed on the extracellular membrane of E. Coli and from there has a an ability to adhere to an antigenic surface.

Function

The part, when expressed correctly, allows for an increased ability for adherence to antigenic surfaces. The antigenic surface can be any common protein provided the synthetic adhesin VHH region is modified to be complimentary. In this case the VHH region has a complimentary binding site of GFP, and so if GFP is fixed to a surface, such as an ELISA plate then any bacteria expressing a complimentary binding region will be better able to adhere to given surface. This can be seen in the image included here but is better seen through our lab work results: https://2020.igem.org/Team:CLS_CLSG_UK/Results

The results from the CLS_CLSG_UK lab work

The results from the original paper published on the use of the SA.

The results we obtained support those found in the original paper.

Design

diagram to show the expression of the SA on the outer membrane

All design details are detailed in the paper 'Programming Controlled Adhesion of E. coli to Target Surfaces, Cells, and Tumors with Synthetic Adhesins' by the Department of Microbial Biotechnology in Spain. They detail how the protein was based off of natural E. coli adhesins: an anchor Beta-barrel module that is embedded in the membrane and an adhesion module bearing an immunoglobulin (Ig) like fold. This synthetic adhesin combines the stable Beta-barrel domain derived from intimin found in EHEC and EPEC E. coli strains. The Ig domain was based on VHH library, a series of variable domains of defined specificity against antigens of interest.

Sequence and Features While this part is not compatible with any of the RFCs we were able to receive the part already embedded into the chromosome of an E. coli from the Department of Microbial Biotechnology who were very co-operative and eager to help.

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 105
  Illegal PstI site found at 401
  Illegal PstI site found at 649
  Illegal PstI site found at 1096
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 105
  Illegal PstI site found at 401
  Illegal PstI site found at 649
  Illegal PstI site found at 1096
  Illegal NotI site found at 2389
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 105
  Illegal BamHI site found at 1983
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 105
  Illegal PstI site found at 401
  Illegal PstI site found at 649
  Illegal PstI site found at 1096
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 105
  Illegal PstI site found at 401
  Illegal PstI site found at 649
  Illegal PstI site found at 1096
  Illegal NgoMIV site found at 2004
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1244
  Illegal BsaI site found at 2063