Difference between revisions of "Part:BBa K3924057"
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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:T--Tsinghua--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''']] | ||
| − | OmpA 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> outer membrane protein A OmpA[1]. The sequence is mainly based on literature we had reviewed and modified by our condon preference system.<br/> | + | OmpA 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> outer membrane protein A OmpA<sup>[1]</sup>. The sequence is mainly based on literature we had reviewed and modified by our condon preference system.<br/> |
==Functional Verification== | ==Functional Verification== | ||
For all candidate secretion peptides, we did codon analysis with our own software tool.(Figure 2) | For all candidate secretion peptides, we did codon analysis with our own software tool.(Figure 2) | ||
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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-OmpA-GFP shows a significant difference with P=0.08. 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-OmpA-GFP shows a significant difference with P=0.08. Therefore, we evaluate this part as a success.<br/> | ||
==Reference== | ==Reference== | ||
| − | [1]Zhao F K, Song Q Z, Wang B B, et al.Secretion of the recombination α-amylase in <i>Escherichia coli</i> and purification by the gram-positive enhancer matrix (GEM) particlesInternational Journal of Biological Macromolecules, 2019,123:91-96.<br/> | + | [1] Zhao F K, Song Q Z, Wang B B, et al.Secretion of the recombination α-amylase in <i>Escherichia coli</i> and purification by the gram-positive enhancer matrix (GEM) particlesInternational Journal of Biological Macromolecules, 2019,123:91-96.<br/> |
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===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K3924057 parameters</partinfo> | <partinfo>BBa_K3924057 parameters</partinfo> | ||
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Latest revision as of 20:56, 21 October 2021
OmpA-GFP
OmpA-GFP
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]
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.
OmpA 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 outer membrane protein A OmpA[1]. The sequence is mainly based on literature we had reviewed and modified by our condon preference system.
Functional Verification
For all candidate secretion peptides, we did codon analysis with our own software tool.(Figure 2)
As for OmpA, 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-OmpA-GFP shows a significant difference with P=0.08. Therefore, we evaluate this part as a success.
Reference
[1] 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.