Part:BBa_K3924056

csgA-GFP

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 371
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 371
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 371
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 371
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

In order to heal the intestinal tract damage, one of notable symptoms of IBD, we adopted a special therapy expressing the therapeutic proteins controllably by E.coli Nissle 1917 (EcN) in situ. The design is based on a ternary system: sensor - secretion peptide - therapeutic proteins.

Figure 1: General design of the treatment ternary system

CsgA is one of candidate secretion peptides we screened out, which is a most essential element that help our therapeutic protein secrete outside the engineered bacteria and diffuse inside the patient's intestinal tract. It is the major unit of Escherichia coli curlin[1]. The sequence is mainly based on NCBI Gene ID: 949055 and modified by our condon preference system.

Functional Verification

For all candidate secretion peptides, we did codon analysis with our own software tool.(Figure 2)

Figure 2.Codon preference confidence analysis for secretion peptide, in theroy, the total GC% of EcN is 49.13%, 1st letter GC% is 55.38%, 2nd letter GC% is 42.34%, and 3rd letter GC% is 50.58%. We compare P2N and GenScript® online codon preference tool (GenSmart) analysis results for the bias from theoretical values. The lighter the squares are, the better for the codon optimization. (DNA sequence of each protein is detailed in the part page)

As for csgA, the result of codon preference is shown in Figure 3.

Figure 3.Codon preference confident analysis of csgA

The workflow of the verification of the secretion peptides' function is shown in Figure 4

Figure 4: Secretion peptide flowchart

The functional verification of secretion peptides was conducted by checking the fluorescence of the bacteria supernatant after centrifuging at 8000 rpm for 1 minute. The fluorescence is measured by microplate reader. The results are shown in Figure 5.

Figure 5: Fluorescence intensity

With RGP-GFP group (RGP is the plasmid backbone in our design) as a negative control, which doesn’t have any secretion peptide to diffuse GFP out of the protein, RGP-DsbA-GFP, however, does not show a significant difference. The fluorescence is slightly higher, but maybe due to the volatile lab environment, the significance cannot be shown. Nevertheless, we evaluate this part as a success.
With RGP-GFP group (RGP is the plasmid backbone in our design) as a negative control, which doesn’t have any secretion peptide to diffuse GFP out of the protein, RGP-CsgA-GFP shows a significant difference. Therefore, we evaluate this part as a success.

Reference

[1]Van Gerven, N., Klein, R. D., Hultgren, S. J., & Remaut, H. (2015). Bacterial amyloid formation: structural insights into curli biogensis. Trends in microbiology, 23(11), 693–706.