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

 
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<p>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.</p>
 
<p>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.</p>
  
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<h2>6His-ProteinA-out</h2>
 
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<p>Part name: BBa_K2509037.</p>
 
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<p>Short description: 6His-ProteinA-out</p>
 
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<p>Schematic diagram:</p>
 
  
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    <img src="https://static.igem.org/mediawiki/2017/f/fa/T-ASTWS-China-p0007.png" alt="" />
 
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  <p>Long description: This part encodes an immunoglobulin G-binding protein A of Staphylococcus aureus with a 6His tag at its N-terminus. Protein A is a 42 kDa surface protein originally found in the cell wall of the bacteria Staphylococcus aureus. It has found use in biochemical research because of its ability to bind immunoglobulins. This part is designed to express at the surface of E.coli by directly being ligated into pET32a Vector or fused with INP or other outer membrane proteins. These E.coli strains that expressed protein A at their surface are thought to have the ability of binding IgG expressing cells or B cells.  </p>
 
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  <p>Source of the part: The main coding region except for the 6His tag of this part is from genomic DNA of Staphylococcus aureus (strain NCTC 8325).</p>
 
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  <p>Design considerations: A 6His tag is fused at the N terminus of this part for easier detection and purification. And two pairs of restriction sites (BamHI+HindIII and NdeI+XhoI) were added on either side of this part so that this part can be ligated with INP or directly ligated into pET32a vector.</p>
 
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  <h2>INP-6His-ProteinA-out</h2>
 
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  <p>Part name: BBa_K2509038</p>
 
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  <p>Short description: INP-6His-ProteinA-out</p>
 
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  <p>Schematic diagram:</p>
 
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    <img src="https://static.igem.org/mediawiki/2017/c/ce/T-ASTWS-China-p0008.png" alt="" />
 
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  <p>Long description: This part encode a fusion protein composed of INP and protein A with 6His tag which is designed to be expressed at the surface of E.coli. We replaced the 6His tag of INP-6His (BBa_K2509019) with an IgG binding protein 6His-protein A (BBa_K2509037) to build this part. After being transferred with this part, the host E.coli strains is considered to have the ability of binding with IgG secreting cells.</p>
 
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  <p>Source of the part: BBa_K2509019 and BBa_K2509037</p>
 
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  <p>Design considerations: The whole fragment of BBa_K2509037 digested with BamHI and HindIII was directly ligated with INP without stop codon to make a ‘sandwich fusion’.</p>
 
  
  

Latest revision as of 03:18, 2 November 2017

INP-2XFlag S

Part name: BBa_K2509020

Short description: INP-2XFlag S

Schematic diagram:

Long description: This part encodes a fusion protein that composed of INP and 2XFlag tag and designed to be expressed at the surface of E.coli. We replaced the 6His tag of INP-6His (BBa_K2509019) with another tag 2XFlag (BBa_K2509007) to build this one. After being transferred with this part, the host E.coli strains is considered to have the ability of binding with anti-Flag magnetic beads.

Source of the part: BBa_K2509019 and BBa_K2509007

Design considerations: we added a stop codon at the end of 2XFlag tag so as to make a C-terminal fusion protein of INP and 2XFlag.

Results:

Dot-Blot result and Western-blot result

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.

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.

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