Part:BBa_K4161100

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 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
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Source

The csg genes are accessed through the E. coli complete genome. The exact sequence we applied comes form 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.