Difference between revisions of "Part:BBa K3924013"
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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:T--Tsinghua--Secretion peptide flowchart.png|center|600px|thumb|'''Figure | + | For all candidate secretion peptides, we did codon analysis with our own software tool.(Figure 2) |
| − | 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 | + | [[Image:T--Tsinghua--Codon preference confidence analysis for secretion peptide.png|center|600px|thumb|'''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) |
| − | [[Image:T--Tsinghua--Fluorescence intensity.png|center|600px|thumb|'''Figure | + | ''']] |
| + | As for pelB, the result of codon preference is shown in Figure 3. | ||
| + | [[Image:T--Tsinghua--Codon preference confident analysis of pelB.png|center|600px|thumb|'''Figure 3.Codon preference confident analysis of pelB''']] | ||
| + | The workflow of the verification of the secretion peptides' function is shown in Figure 4 | ||
| + | [[Image:T--Tsinghua--Secretion peptide flowchart.png|center|600px|thumb|'''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.<br/> | ||
| + | [[Image:T--Tsinghua--Fluorescence intensity.png|center|600px|thumb|'''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.<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-PelB-GFP shows a significant difference with P=0.09. Therefore, 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-PelB-GFP shows a significant difference with P=0.09. Therefore, we evaluate this part as a success.<br/> | ||
==Reference== | ==Reference== | ||
Revision as of 20:22, 21 October 2021
PelB
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: PelB
Base Pairs: 69
Origin: Erwinia carotovora
Properties: Leader sequence of Erwinia carotovora endo-pectin lyase PelB
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.
PelB 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 leader sequence of Erwinia carotovora endo-pectin lyase PelB[4]. 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)
As for pelB, the result of codon preference is shown in Figure 3.
The workflow of the verification of the secretion peptides' function is shown in Figure 4
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.
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-PelB-GFP shows a significant difference with P=0.09. Therefore, 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.