Difference between revisions of "Part:BBa K3924010"

Revision as of 07:30, 21 October 2021

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

Figure 2: 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 3.

Figure 3: 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.

@@ Line 75: / Line 75: @@
 	After getting the codon-optimized sequence for <i>E. coli</i>, we synthesized the sequence by company, and linked them to a GFP element by using HiFi Assembly.<br/>
 ==Functional Verification==
-[[Image:Secretion peptide flowchart.png|'''Figure 2: Secretion peptide flowchart''']]
+[[Image:Secretion peptide flowchart.png|center|600px|thumb|'''Figure 2: 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 3.<br/>
-[[Image:Fluorescence intensity.png|'''Figure 3: Fluorescence intensity''']]
+[[Image:Fluorescence intensity.png|center|600px|thumb|'''Figure 3: 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.<br/>
 ==Reference==