Difference between revisions of "Part:BBa K3924010"

 
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<partinfo>BBa_K3924010 SequenceAndFeatures</partinfo>
 
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==Profile==
 +
Name: DsbA<br/>
 +
Base Pairs: 60<br/>
 +
Origin: <i>Escherichia coli</i><br/>
 +
Properties: Siganl peptide of <i>Escherichia coli</i> thiol:disulfide oxidoreductase DsbA<br/>
 +
==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/>
 +
[[Image:General design of the treatment ternary system.png|center|600px|thumb|'''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 <i>Escherichia coli</i> thiol:disulfide oxidoreductase DsbA[1]. The sequence is mainly based on literature we had reviewed and modified by our condon preference system.<br/>
 +
==Design and Construction==
 +
According to literature research we chose 7 candidate secretion peptides and did codon analysis with our own software tool.<br/>
 +
Table 1. List of candidate therapeutic proteins
 +
<table border="1">
 +
  <tr>
 +
    <th>Part Name</th>
 +
    <th>Element Name</th>
 +
    <th>Origin</th>
 +
    <th>Reference</th>
 +
  </tr>
 +
  <tr>
 +
    <td>BBa_K3924010</td>
 +
    <td>DsbA</td>
 +
    <td><i>E. coli</i> periplasmic space</td>
 +
    <td>[1]</td>
 +
  </tr>
 +
  <tr>
 +
    <td>BBa_K3924011</td>
 +
    <td>CsgA</td>
 +
    <td><i>E. coli</i> biofilm matrix</td>
 +
    <td>[2]</td>
 +
  </tr>
 +
  <tr>
 +
    <td>BBa_K3924012</td>
 +
    <td>OmpA</td>
 +
    <td><i>E. coli</i> outer membrane</td>
 +
    <td>[3]</td>
 +
  </tr>
 +
  <tr>
 +
    <td>BBa_K3924013</td>
 +
    <td>PelB</td>
 +
    <td><i>Erwinia carotovora</i> periplasmic space</td>
 +
    <td>[4]</td>
 +
  </tr>
 +
  <tr>
 +
    <td>BBa_K3924014</td>
 +
    <td>PhoA</td>
 +
    <td><i>E. coli</i> periplasmic space</td>
 +
    <td>[5]</td>
 +
  </tr>
 +
  <tr>
 +
    <td>BBa_K3924015</td>
 +
    <td>STⅡ</td>
 +
    <td><i>E. coli</i> extracellular peptide toxin</td>
 +
    <td>[6]</td>
 +
  </tr>
 +
  <tr>
 +
    <td>BBa_K3924016</td>
 +
    <td>TorA</td>
 +
    <td><i>E. coli</i> periplasmic space</td>
 +
    <td>[7]</td>
 +
  </tr>
 +
</table>
 +
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''']]
 +
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''']]
 +
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==
 +
[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.<br/>
 +
[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.<br/>
 +
[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.<br/>
 +
[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<br/>
 +
[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.<br/>
 +
[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.<br/>
 +
[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.<br/>
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K3924010 parameters</partinfo>
 
<partinfo>BBa_K3924010 parameters</partinfo>
 
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Revision as of 07:19, 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.