Part:BBa_K3924010

DsbA

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]

Profile

Name: DsbA
Base Pairs: 60
Origin: Escherichia coli
Properties: Siganl peptide of Escherichia coli thiol:disulfide oxidoreductase DsbA

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

DsbA 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 a siganl peptide of Escherichia coli thiol:disulfide oxidoreductase DsbA[1]. The sequence is mainly based on literature we had reviewed and modified by our condon preference system.

Design and Construction

According to literature research we chose 7 candidate secretion peptides and did codon analysis with our own software tool.
Table 1. List of candidate therapeutic proteins

Part Name	Element Name	Origin	Reference
BBa_K3924010	DsbA	E. coli periplasmic space	[1]
BBa_K3924011	CsgA	E. coli biofilm matrix	[2]
BBa_K3924012	OmpA	E. coli outer membrane	[3]
BBa_K3924013	PelB	Erwinia carotovora periplasmic space	[4]
BBa_K3924014	PhoA	E. coli periplasmic space	[5]
BBa_K3924015	STⅡ	E. coli extracellular peptide toxin	[6]
BBa_K3924016	TorA	E. coli periplasmic space	[7]

After getting the codon-optimized sequence for E. coli, we synthesized the sequence by company, and linked them to a GFP element by using HiFi Assembly.

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 DsbA, the result of codon preference is shown in Figure 3.

Figure 3.Codon preference confident analysis of DsbA

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.

Reference

[1] Zhou Y Z, Liu P, Gan Y T, et al.Enhancing full-length antibody production by signal peptide engineering.Microbial Cell Factories, 2016,15(1):1-11.
[2] 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.
[3] Zhao F K, Song Q Z, Wang B B, et al.Secretion of the recombination α-amylase in Escherichia coli and purification by the gram-positive enhancer matrix (GEM) particlesInternational Journal of Biological Macromolecules, 2019,123:91-96.
[4] Sriwidodo S, Subroto T, Maksum I, et al.Optimization of secreted recombinant human epidermal growth factor production using pectate lyase B from Escherichia coli BL21(DE3) by central composite design and its production in high cell density culture
[5] Mohajeri A, Abdolalizadeh J, Pilehvar-Soltanahmadi Y, et al.Expression and secretion of endostar protein by Escherichia coli: optimization of culture conditions using the response surface methodology Molecular Biotechnology, 2016,58(10):634-647.
[6] Lu C, Zhao H, Zou W Y, et al.Secretion expression of recombinate human interferon α-2b by Escherichia coli Journal of Biology, 2011,28(3):58-62.
[7] Guerrero Montero I, Richards K L, Jawara C, et al.Escherichia coli “TatExpress” strains export several g/L human growth hormone to the periplasm by the Tat pathway Biotechnology and Bioengineering, 2019,116(12):3282-3291.