Difference between revisions of "Team:ASTWS-China/Basic Part"

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               <h2 class="header">COMMON PAGE</h2>
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               <h2 class="header">INP with 6His tag:</h2>
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               <p>
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                Our idea of project comes from Hybridoma technology. We found some disadvantage about the old technology. In order to improve this technology, we started this project. We also want to know how most of people thinks about Hybridoma technology and our new approach. So we make a questioner to collect others view about our project.
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               <p>Part name: BBa_K2509019.</p>
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               </p>
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               <p>Short description: INP with 6His tag</p>
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              <p>Schematic diagrams (design):</p>
 
               <div class="images">
 
               <div class="images">
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                 <img src="https://static.igem.org/mediawiki/2017/e/e8/T-ASTWS-China-00001.jpeg" alt="" />
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                 <img src="https://static.igem.org/mediawiki/2017/d/d5/T-ASTWS-China-p0003.png" alt="" />
 
               </div>
 
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              <p>Long description: This is an improved version of BBa_K584021 composed of 6His tag and partial sequence of INP. The ice nucleation protein (INP) is a glycosyl phosphatidylinositol anchored outer membrane protein found in Pseudomonas syringae and the 6His tag is a commonly used small amino acid motiff that consists of six histidine residues. We replaced the coding sequence from 222aa to 1061aa of INP with a 6His tag flanked by BamHI and HindIII sites to make this part. Further we added an NdeI site before the start codon and an XhoI site after the final stop codon in order that it could be easily ligated into pET expression vectors. This part can be used in the surface display of recombinant proteins or small tags on Escherichia coli by replacing 6His tag with a target protein with or without a stop codon.</p>
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              <p>Source of the part: The partial coding region of INP is from BBa_K584021, and the 6His tag is a commonly used small amino acid motiff that consists of six histidine residues. The whole parts is synthesized by GenScript Corporation.</p>
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              <p>Design considerations: First we added an NdeI site before the start codon and an XhoI site after the final stop codon in order that it could be easily ligated into pET32 vector, secondary we added BamHI and HindIII sites at two sides of the 6His tag so that this small tag could be easily replaced by other proteins, and also we added a stop codon at the end of the 6His tag so that this part can only actually express an N part of INP fused with 6His tag at the C-terminus.</p>
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              <p>Results</p>
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              <p>Dot-Blot result and Western-blot result</p>
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              <div class="images">
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                <img src="https://static.igem.org/mediawiki/2017/7/73/T-ASTWS-China-p0010.jpg" alt="" />
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              </div>
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              <p>Conclusion: This part was expressed correctly in E.coli.</p>
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              <p>Immunofluorescence (IF) results</p>
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              <div class="images">
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                <img src="https://static.igem.org/mediawiki/2017/8/88/T-ASTWS-China-p0011.jpg" alt="" />
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              </div>
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              <p>Conclusion: The 6His tag was expressed at the surface of E.coli strain as designed.</p>
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              <p>Binding results of E.coli strains and NTA beads</p>
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              <div class="images">
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                <img src="https://static.igem.org/mediawiki/2017/7/73/T-ASTWS-China-p0012.jpg" alt="" />
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              </div>
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              <p>Conclusion: The 6His tag was expressed at the surface of E.coli strain as designed, and the modified host can bind with NTA beads.</p>
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              <p>References:</p>
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              <p>
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                1. Fan LH, Liu N, Yu MR, Yang ST, Chen HL. Cell surface display of carbonic anhydrase on Escherichia coli using ice nucleation protein for CO₂ sequestration. Biotechnol Bioeng. 2011 Dec; 108(12):2853-64.
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              </p>
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              <p>
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                2. Eui-Joong Kim and Seung-Ku Yoo. Cell surface display of CD8 ecto domain on Escherichia coli using ice nucleation protein. Biotechnology Techniques. 1998 Mar; 12(3):197–201.
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              </p>
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              <p>
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                3. Kim EJ, Yoo SK.Cell surface display of hepatitis B virus surface antigen by using Pseudomonas syringae ice nucleation protein. Lett Appl Microbiol. 1999 Nov; 29(5):292-7.
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              </p>
 
               <p>
 
               <p>
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                 To know the opinion from normal people, we started a questionnaire on WeChat.
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                 4. Chungjatupornchai W, Fa-aroonsawat S. Translocation of green fluorescent protein to cyanobacterial periplasm using ice nucleation protein. J Microbiol. 2009 Apr; 47(2):187-92.
 
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Revision as of 12:59, 30 October 2017

INP with 6His tag:

Part name: BBa_K2509019.

Short description: INP with 6His tag

Schematic diagrams (design):

Long description: This is an improved version of BBa_K584021 composed of 6His tag and partial sequence of INP. The ice nucleation protein (INP) is a glycosyl phosphatidylinositol anchored outer membrane protein found in Pseudomonas syringae and the 6His tag is a commonly used small amino acid motiff that consists of six histidine residues. We replaced the coding sequence from 222aa to 1061aa of INP with a 6His tag flanked by BamHI and HindIII sites to make this part. Further we added an NdeI site before the start codon and an XhoI site after the final stop codon in order that it could be easily ligated into pET expression vectors. This part can be used in the surface display of recombinant proteins or small tags on Escherichia coli by replacing 6His tag with a target protein with or without a stop codon.

Source of the part: The partial coding region of INP is from BBa_K584021, and the 6His tag is a commonly used small amino acid motiff that consists of six histidine residues. The whole parts is synthesized by GenScript Corporation.

Design considerations: First we added an NdeI site before the start codon and an XhoI site after the final stop codon in order that it could be easily ligated into pET32 vector, secondary we added BamHI and HindIII sites at two sides of the 6His tag so that this small tag could be easily replaced by other proteins, and also we added a stop codon at the end of the 6His tag so that this part can only actually express an N part of INP fused with 6His tag at the C-terminus.

Results

Dot-Blot result and Western-blot result

Conclusion: This part was expressed correctly in E.coli.

Immunofluorescence (IF) results

Conclusion: The 6His tag was expressed at the surface of E.coli strain as designed.

Binding results of E.coli strains and NTA beads

Conclusion: The 6His tag was expressed at the surface of E.coli strain as designed, and the modified host can bind with NTA beads.

References:

1. Fan LH, Liu N, Yu MR, Yang ST, Chen HL. Cell surface display of carbonic anhydrase on Escherichia coli using ice nucleation protein for CO₂ sequestration. Biotechnol Bioeng. 2011 Dec; 108(12):2853-64.

2. Eui-Joong Kim and Seung-Ku Yoo. Cell surface display of CD8 ecto domain on Escherichia coli using ice nucleation protein. Biotechnology Techniques. 1998 Mar; 12(3):197–201.

3. Kim EJ, Yoo SK.Cell surface display of hepatitis B virus surface antigen by using Pseudomonas syringae ice nucleation protein. Lett Appl Microbiol. 1999 Nov; 29(5):292-7.

4. Chungjatupornchai W, Fa-aroonsawat S. Translocation of green fluorescent protein to cyanobacterial periplasm using ice nucleation protein. J Microbiol. 2009 Apr; 47(2):187-92.

Copyright © 2017 Team:ASTWS-China All Rights Reserved.