Difference between revisions of "Part:BBa K2552005"
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Ompa-Oligo peptide T-1 part uses Ompa system to display oligo peptide T-1 on the surface of E.coli cells.
 
Ompa-Oligo peptide T-1 part uses Ompa system to display oligo peptide T-1 on the surface of E.coli cells.
  
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===Usage and Biology===
 
===Usage and Biology===
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It's an improvment of ompA(BBa_K).
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It’s an improvement of OmpA([https://parts.igem.org/Part:BBa_K1489002 BBa_K1489002]
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). OmpA is widely utilized to anchor otherwise soluble proteins in the outer cell membrane of bacteria like E.coli. Thus, OmpA functions as a carrier that carry different protein or domain to the surface of bacterial cells.
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Considering more and more teams are focusing on cancer targeting, we added a 15-AA oligo peptide to the C-terminal of OmpA to give it the ability to target cancer. The oligo peptide has the ability to bind to Thomsen–Friedenreich antigen (T antigen) that exist on some kinds of cancer cells(like colorectal cancer) by a mechanism similar to antigen-antibody binding reaction. On the other hand, the oligo peptide is so short that its influence on the function of OmpA is limited. That is, when OmpA is carrying other peptides or protein domains at the C terminal, there is little chance that the peptide will cause adverse effects such as misfolding.
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===Experiment design===
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Experiments have been conducted by Team SJTU-BioX-Shanghai.  We used cell-climbing cover glasses as the solid basement on which the cell-bacteria adhesion experiment was carried out. Cell line HT29(human colorectal cancer cell) was chosen for the experiment, which grew over the glasses. We certificate the expression of T antigen by FACS previously. After cell HT29 grew and covered the glasses， we mixed it with E.coli BL21(DE3) cultures which expressed OmpA-peptide fusion protein (egfp was expressed as reporter gene ). The mixture glasses were incubated at 4°C for hours(results of 12h are shown) after which cells were cleaned with normal saline to wash away unconjugated bacteria. These cover glasses were carefully observed via the fluorescent microscope.
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===Protein structure modeling===
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We use Swiss Model to predict the structure of our OmpA-peptide fusion protein. The result shows us that the oligo peptide don’t influence the correct folding of the β-barrel of OmpA.
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[[Image:K25520005.png|centre|500px]]
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===Experimental results===
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[[Image:ompa-tp1.png|left|200px]]<br/>[[Image:ompa-egfp.png|left|200px]]<br/>
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Fig1. The results of our experiments.The first image showed that some bacteria with OmpA-pep1 have the ability to attach to the surface of HT29, indicating that the targeting is effective.The second picture is the result of control(OmpA without oligo peptide). Nearly no bacteria is attached to the surface of the cell.<br/>
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 23:00, 17 October 2018


Ompa-Oligo peptide T-1

Ompa-Oligo peptide T-1 part uses Ompa system to display oligo peptide T-1 on the surface of E.coli cells.

Usage and Biology

It’s an improvement of OmpA(BBa_K1489002 ). OmpA is widely utilized to anchor otherwise soluble proteins in the outer cell membrane of bacteria like E.coli. Thus, OmpA functions as a carrier that carry different protein or domain to the surface of bacterial cells.

Considering more and more teams are focusing on cancer targeting, we added a 15-AA oligo peptide to the C-terminal of OmpA to give it the ability to target cancer. The oligo peptide has the ability to bind to Thomsen–Friedenreich antigen (T antigen) that exist on some kinds of cancer cells(like colorectal cancer) by a mechanism similar to antigen-antibody binding reaction. On the other hand, the oligo peptide is so short that its influence on the function of OmpA is limited. That is, when OmpA is carrying other peptides or protein domains at the C terminal, there is little chance that the peptide will cause adverse effects such as misfolding.

Experiment design

Experiments have been conducted by Team SJTU-BioX-Shanghai. We used cell-climbing cover glasses as the solid basement on which the cell-bacteria adhesion experiment was carried out. Cell line HT29(human colorectal cancer cell) was chosen for the experiment, which grew over the glasses. We certificate the expression of T antigen by FACS previously. After cell HT29 grew and covered the glasses， we mixed it with E.coli BL21(DE3) cultures which expressed OmpA-peptide fusion protein (egfp was expressed as reporter gene ). The mixture glasses were incubated at 4°C for hours(results of 12h are shown) after which cells were cleaned with normal saline to wash away unconjugated bacteria. These cover glasses were carefully observed via the fluorescent microscope.

Protein structure modeling

We use Swiss Model to predict the structure of our OmpA-peptide fusion protein. The result shows us that the oligo peptide don’t influence the correct folding of the β-barrel of OmpA.

K25520005.png

Experimental results

Ompa-tp1.png

Ompa-egfp.png





















Fig1. The results of our experiments.The first image showed that some bacteria with OmpA-pep1 have the ability to attach to the surface of HT29, indicating that the targeting is effective.The second picture is the result of control(OmpA without oligo peptide). Nearly no bacteria is attached to the surface of the cell.
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 site found at 485