Difference between revisions of "Part:BBa K3924000"

 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3924000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3924000 SequenceAndFeatures</partinfo>
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+
==Profile==
 +
Name: TFF1<br/>
 +
Base Pairs: 369bp<br/>
 +
Origin: <i>Homo sapiens</i><br/>
 +
Properties: One of human cytokine from The trefoil factor (TFF) family secreted by mucus-producing cells. <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 E.coli Nissle 1917 (EcN) in situ. The design is based on a ternary system: sensor - secretion peptide - therapeutic proteins.
 +
[[Image: General design of the treatment ternary system.png|'''Fig.1 General design of the treatment ternary system''']]
 +
TFF1 is one of the candidate therapeutic proteins we screened out to treat IBD, which is the effector element in the ternary system. TFF facilitate a significant role not only in mucosal repair but also in protecting mucous epithelia from a variety of insults in the gastrointestinal tract. The potential mechanisms to treat IBD involving anti-apoptotic properties, migration and invasion, angiogenesis, and interaction with mucins.
 +
==Design and Construction==
 +
According to literature research we chose 10 candidate proteins for IBD treatment.
 +
Table 1. List of candidate therapeutic proteins
 +
Table 1. List of candidate therapeutic proteins
 +
<table border="1">
 +
  <tr>
 +
    <th>Part Name</th>
 +
    <th>Element Name</th>
 +
    <th>Reference</th>
 +
  </tr>
 +
  <tr>
 +
    <td> BBa_K3924000<br/>BBa_K3924001<br/>BBa_K3924002</td>
 +
    <td>TFF1/2/3</td>
 +
    <td>[1][2]</td>
 +
  </tr>
 +
  <tr>
 +
    <td> BBa_K3924003</td>
 +
    <td>Chromofungin(CHR: CHGA47-66)</td>
 +
    <td>[3]</td>
 +
  </tr>
 +
  <tr>
 +
    <td> BBa_K3924004</td>
 +
    <td>IL10</td>
 +
    <td>[4]</td>
 +
  </tr>
 +
  <tr>
 +
    <td> BBa_K3924005</td>
 +
    <td>Defensin-5(HD5)</td>
 +
    <td>[5]</td>
 +
  </tr>
 +
  <tr>
 +
    <td> BBa_K3924006</td>
 +
    <td>GLP2</td>
 +
    <td>[6]</td>
 +
  </tr>
 +
  <tr>
 +
    <td> BBa_K3924007</td>
 +
    <td>Fgl2</td>
 +
    <td>[7]</td>
 +
  </tr>
 +
  <tr>
 +
    <td> BBa_K3924008</td>
 +
    <td>SOCS1-KIR</td>
 +
    <td>[3]</td>
 +
  </tr>
 +
<tr>
 +
    <td> BBa_K3924009</td>
 +
    <td>tkip</td>
 +
    <td>[3]</td>
 +
  </tr>
 +
</table>
 +
The sequence of TFF1 is acquired from Gene database (Gene ID: 7031). The sequences were synthesized on pMV plasmids by biological companies and cloned into pEGFP & RGP-TMAR plasmids by ourselves for the following experiments. PCR technique and HiFi assembly method were frequently utilized. Eventually, we have successfully constructed the plasmids RGP-csgA-TFF1 as the best carrier of our treatment protein. The plasmids were amplified inside E. coli DH5α and imported into E. coli Nissle through electrotransformation. The protein expression of the TFF1 was tested using SDS-PAGE and western blot.
 +
==Functional Verification==
 +
[[Image: The scheme of the proof-of-concept for therapeutic proteins.png|'''Fig 2. The scheme of the proof-of-concept for therapeutic proteins.''']]
 +
All of these proteins are worth studying, but we only chose a few proteins as a proof of concept in our actual wet lab experiments because of the time limit and the high expense of gene synthesis.
 +
For TFF1 we did codon analysis with our own software tool, and the results are shown in Fig.
 +
==Reference==
 +
[1] Aamann, L., Vestergaard, E. M., & Grønbæk, H. (2014). Trefoil factors in inflammatory bowel disease. World journal of gastroenterology, 20(12), 3223–3230.
 +
[2] Praveschotinunt, P., Duraj-Thatte, A.M., Gelfat, I. et al. Engineered E. coli Nissle 1917 for the delivery of matrix-tethered therapeutic domains to the gut. Nat Commun 10, 5580 (2019)..
  
 
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Revision as of 06:42, 21 October 2021


TFF1

aaa

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 13

Profile

Name: TFF1
Base Pairs: 369bp
Origin: Homo sapiens
Properties: One of human cytokine from The trefoil factor (TFF) family secreted by mucus-producing cells.

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. Fig.1 General design of the treatment ternary system TFF1 is one of the candidate therapeutic proteins we screened out to treat IBD, which is the effector element in the ternary system. TFF facilitate a significant role not only in mucosal repair but also in protecting mucous epithelia from a variety of insults in the gastrointestinal tract. The potential mechanisms to treat IBD involving anti-apoptotic properties, migration and invasion, angiogenesis, and interaction with mucins.

Design and Construction

According to literature research we chose 10 candidate proteins for IBD treatment. Table 1. List of candidate therapeutic proteins Table 1. List of candidate therapeutic proteins

Part Name Element Name Reference
BBa_K3924000
BBa_K3924001
BBa_K3924002		 TFF1/2/3 [1][2]
BBa_K3924003 Chromofungin(CHR: CHGA47-66) [3]
BBa_K3924004 IL10 [4]
BBa_K3924005 Defensin-5(HD5) [5]
BBa_K3924006 GLP2 [6]
BBa_K3924007 Fgl2 [7]
BBa_K3924008 SOCS1-KIR [3]
BBa_K3924009 tkip [3]

The sequence of TFF1 is acquired from Gene database (Gene ID: 7031). The sequences were synthesized on pMV plasmids by biological companies and cloned into pEGFP & RGP-TMAR plasmids by ourselves for the following experiments. PCR technique and HiFi assembly method were frequently utilized. Eventually, we have successfully constructed the plasmids RGP-csgA-TFF1 as the best carrier of our treatment protein. The plasmids were amplified inside E. coli DH5α and imported into E. coli Nissle through electrotransformation. The protein expression of the TFF1 was tested using SDS-PAGE and western blot.

Functional Verification

Fig 2. The scheme of the proof-of-concept for therapeutic proteins. All of these proteins are worth studying, but we only chose a few proteins as a proof of concept in our actual wet lab experiments because of the time limit and the high expense of gene synthesis. For TFF1 we did codon analysis with our own software tool, and the results are shown in Fig.

Reference

[1] Aamann, L., Vestergaard, E. M., & Grønbæk, H. (2014). Trefoil factors in inflammatory bowel disease. World journal of gastroenterology, 20(12), 3223–3230. [2] Praveschotinunt, P., Duraj-Thatte, A.M., Gelfat, I. et al. Engineered E. coli Nissle 1917 for the delivery of matrix-tethered therapeutic domains to the gut. Nat Commun 10, 5580 (2019)..