Part:BBa_K4161003

Csg surface display system on E. coli Nissle 1917

We intend to display VHH (in this example, JMK-H2) on the surface of E. coli Nissle 1917 through curli fibers and seliver it to intestines to treat shigella infection. The display system is mainly based on works of Gelfat et al. (2021) The production of culi fibers is encoded by csg genes. The csgDEFG genes encodes proteins that secrete curli fiber units - csgA & csgB, while csgC protein prevents fiber formation in the bacterium. As shown in the plasmid, the VHH (JMK-H2) is conjugated with csgA protein by flexible linker FE36 containing E-tag, and will be transported to the extracellular space, then displayed along with csgA protein. Apart from the E tag, the VHH is also marked with 3xFlag tag at its C terminal. pUC19 is chosen as the plasmid backbone, and the pTlpA (temperature responsive promotor) was added to ensure normal expression of csg genes in gut environment.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2611
  Illegal XbaI site found at 82
  Illegal SpeI site found at 1795
  Illegal PstI site found at 13
  Illegal PstI site found at 1064
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2611
  Illegal SpeI site found at 1795
  Illegal PstI site found at 13
  Illegal PstI site found at 1064
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2611
  Illegal BamHI site found at 1
  Illegal XhoI site found at 1233
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2611
  Illegal XbaI site found at 82
  Illegal SpeI site found at 1795
  Illegal PstI site found at 13
  Illegal PstI site found at 1064
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2611
  Illegal XbaI site found at 82
  Illegal SpeI site found at 1795
  Illegal PstI site found at 13
  Illegal PstI site found at 1064
  Illegal AgeI site found at 2908
  
• 1000
  COMPATIBLE WITH RFC[1000]

Source

The original sequence that we borrowed can be accessed through the plasmid pM1s3ATScsg-Etag (https://www.addgene.org/137946/)

Reference

Gelfat, I., Mitragotri, S., Joshi, N., Mooney, D., & Leong, J. (2021) Engineering E. coli Nissle 1917 to advance and facilitate its use in biomedical applications. Harvard University.