Difference between revisions of "Part:BBa K3924016"
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==Usage and Biology== | ==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 <i>E.coli Nissle 1917</i> (EcN) in situ. The design is based on a ternary system: sensor - secretion peptide - therapeutic proteins.<br/> | 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 <i>E.coli Nissle 1917</i> (EcN) in situ. The design is based on a ternary system: sensor - secretion peptide - therapeutic proteins.<br/> | ||
| − | [[Image:General design of the treatment ternary system.png|center|600px|thumb|'''Figure 1: General design of the treatment ternary system''']] | + | [[Image:T--Tsinghua--General design of the treatment ternary system.png|center|600px|thumb|'''Figure 1: General design of the treatment ternary system''']] |
TorA 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 signal peptide of <i>Escherichia coli</i> trimethylamine N-oxide reductase 1[7]. The sequence is mainly based on literature we had reviewed and modified by our condon preference system.<br/> | TorA 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 signal peptide of <i>Escherichia coli</i> trimethylamine N-oxide reductase 1[7]. The sequence is mainly based on literature we had reviewed and modified by our condon preference system.<br/> | ||
==Design and Construction== | ==Design and Construction== | ||
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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/> | 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== | ==Functional Verification== | ||
| − | [[Image:Secretion peptide flowchart.png|center|600px|thumb|'''Figure 2: Secretion peptide flowchart''']] | + | [[Image:T--Tsinghua--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/> | 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|center|600px|thumb|'''Figure 3: Fluorescence intensity''']] | + | [[Image:T--Tsinghua--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-TorA-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/> | 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-TorA-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== | ==Reference== | ||
Revision as of 19:09, 21 October 2021
TorA
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Profile
Name: TorA
Base Pairs: 132
Origin: Escherichia coli
Properties: Signal peptide of Escherichia coli trimethylamine N-oxide reductase 1
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.
TorA 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 signal peptide of Escherichia coli trimethylamine N-oxide reductase 1[7]. 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
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.
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-TorA-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.