Difference between revisions of "Team:SHSBNU China/Improve"

 
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<p style="font-family: Serif;font-size: 3.5vw; font-weight: bold;text-align: center;margin: 0.5vw 0vw 0.5vw 0vw">Improve</p>
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<h6 id="menu_intro">Improve</h6>
 
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#I">Overview</a>
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#I">Demonstration</a>
 
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#ID">Improvement Details</a>
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#VI">Using sfGFP-SpyCatcher</a>
 
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            <a class="trd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#a">CsgA Improvement</a>
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#VII">Verification - Using SpyCatcher-CotA</a>
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#C">Conclusion</a>
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Project">Project</a>
 
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            <a class="trd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#b">CotA Improvement</a>
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<h2 id="I">Overview</h2>
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<h2 id="I">I. Overview</h2>
 
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<img class="pictures" id = "21000" src="https://static.igem.org/mediawiki/2018/b/b9/T--SHSBNU_China--21000.png"/>
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<p class="pic_text">Fig1.1: CsgA-SpyTag</p>
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<p class="text">
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    CsgA is a amyloid protein which is a dominant component of the biofilms of E. coli. We fused a functional peptide domain SpyTag with gene CsgA (previous part: BBa_K1583000), forming CsgA-SpyTag (Improved Part: BBa_K2684006). By utilizing the steady isopeptide bond combination ability of the SpyTag-SpyCatcher system , it is possible to fix different SpyChatcher-fusing proteins, like enzymes or receptors, to the biofilm to introduce a variety of artificial functions.
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(Team: Peking, 2016)
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<img class="pictures" id = "Improve_1" src="https://static.igem.org/mediawiki/2018/b/bb/T--SHSBNU_China--Improve_1.jpg"/>
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<p class="pic_text">Fig1.2: SpyTag and SpyCatcher System</p>
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<p><img src="https://static.igem.org/mediawiki/2018/b/b9/T--SHSBNU_China--21000.png" style="width:75%"/></image></p>
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<h2 id="VI">II. Verification - Using sfGFP-SpyCatcher</h2>
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    Previously, there was only CsgA part uploaded in iGEM parts website. Our Team improved part <a href="http://parts.igem.org/Part:BBa_K1583000">Part BBa_K1583000</a> by adding a SpyTag sequence. It fused to gene <em>csgA</em>, enabling CotA laccase to be fixed onto the biofilm to form a covalent bond--SpyTag-SpyCatcher.
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We used sfGFP-SpyCatcher as indicator to test whether the addition of SpyTag peptide could confer the artificial functions to the biofilm, such as covalent immobilization of SpyCatcher-fusion proteins. </p>
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<a href="https://2018.igem.org/Team:SHSBNU_China/Protocol#SSS">
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Protocol for SpyTag-SpyCatcher system verification
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</a>
 
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We tested our improved part BBa_K2684006 and the previous part BBa_K1583000 with the same protein expression system settings (E. coli BL21, pET28a vector, and the same induction and expression protocol).</p>
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<img class="pictures" id = "Improve_2" src="https://static.igem.org/mediawiki/2018/9/90/T--SHSBNU_China--Improve_2.png"/>
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The decrease of florescence of the supernatant indicates the immobilization of sfGFP-SpyCatcher, and there’s a significant difference between the two groups.</p>
 
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<h2 id="ID">Improvement Details</h2>
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        <h3 id="a">CsgA Improvement</h3>
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<h2 id="VII">III. Verification - Using SpyCatcher-CotA</h2>
 
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<img class="pictures" id = "Improve_3" src="https://static.igem.org/mediawiki/2018/7/7c/T--SHSBNU_China--Improve_3.png"/>
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The immobilization of SpyCatcher-CotA fusion protein to the CsgA-SpyTag biofilm was tested, and a commercial laccase activity assay kits was used to measure the laccase activity. </p>
 
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By using sfGFP-SpyCatcher protein, we tested the binding efficiency of the covalence SpyTag-SpyCatcher since sfGFP is a non-toxic and common used flurorescent protein. Gene <em>csgA</em> on the plasmid of pET28a was transferred into ΔMG1655 as control group. Gene <em>csgA – spytag</em> on the plasmid of pET28a was transferred into ΔMG1655 as experiment. The experiment was divided into 4 groups. The bateria in the Reaction Stock had neither <em>csgA</em> nor <em>SpyTag</em> in neither genome nor plasmid; the ones in Group 1 had <em>csgA</em> in the genome; the ones in Group 2 had <em>sfGFP</em> in the plasmid; the ones in Group 3 had <em>csgA-SpyTag</em> in the genome and the plasmid. The treatment of the experiment is followed by the procedure below:
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<a href="https://2018.igem.org/Team:SHSBNU_China/Protocol#BXL">
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Protocol for Biofilm x Laccase
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</a>
 
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The difference between our improved part and the previous part is significant. Thus, we proved that SpyCatcher-CotA can be attached to the biofilm containing functional peptide SpyTag. </p>
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<img class="pictures" id = "21002" src="https://static.igem.org/mediawiki/2018/5/57/T--SHSBNU_China--21002.jpg"/></image>
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<p class="pic_text">The liquid after centrifugation was extracted and measured fluorescence. </p>
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<h2 id="C">IV. Conclusion</h2>
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  As can be seen from the result, the experiment group showed the most decrease of sfGFP-SpyCatcher protein. The difference between the control group and experiment group was significant. Thus we could confirm our CsgA-SpyTag system was functional.</p>
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To conclude, we introduce a new ability to the previous CsgA part, which is capturing all fusion protein with SpyCatcher domain, by fusing SpyTag peptide to CsgA coding sequence. We verified the protein immobilization function of our improved part using sfGFP-SpyCatcher and SpyCatcher-CotA, and proved that SpyCatcher fusion protein is still functional after fixed to the biofilm.
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                      Part BBa_K2684000 was improved from previous part <a href="http://parts.igem.org/Part:BBa_K1336002">BBa_K1336002</a>. Our team made a point mutation to eliminate the EcoR1 cutting site so that we can meet the standard of RFC10, a commonly-used standard for interchangeable parts which are based on idempotent assembly. We tested the enzyme activity of our part based on the weight of our sample. Formula: laccase activity (nmol/min/g) = ΔA /ε(ABTS mmolar extinction coefficient) / d * V (total volume of reaction) / V (volume of sample in the reaction, 0.025mL) / W (sample mass, g) * V (extracted liquid added, 1mL) / T (reaction time, 3 minutes) = 130 *ΔA / W. The result is shown as following: </p>
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                    <p><img src="https://static.igem.org/mediawiki/2018/4/42/T--SHSBNU_China--Parts_Regis.png" style="width:50%"/></image></p>
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                    <p class="text">
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<strong>Reference:</strong>
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                      The improvement is valid since it was normally produced.
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</p>
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                    </p>
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<p class="text">
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“Uranium Reaper.” <i>Uranium Reaper - Team: Peking</i>, 2016.igem.org/Team:Peking.
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Latest revision as of 03:55, 18 October 2018

Improvements

Improve

I. Overview

Fig1.1: CsgA-SpyTag

CsgA is a amyloid protein which is a dominant component of the biofilms of E. coli. We fused a functional peptide domain SpyTag with gene CsgA (previous part: BBa_K1583000), forming CsgA-SpyTag (Improved Part: BBa_K2684006). By utilizing the steady isopeptide bond combination ability of the SpyTag-SpyCatcher system , it is possible to fix different SpyChatcher-fusing proteins, like enzymes or receptors, to the biofilm to introduce a variety of artificial functions.

(Team: Peking, 2016)

Fig1.2: SpyTag and SpyCatcher System

II. Verification - Using sfGFP-SpyCatcher

We used sfGFP-SpyCatcher as indicator to test whether the addition of SpyTag peptide could confer the artificial functions to the biofilm, such as covalent immobilization of SpyCatcher-fusion proteins.

Protocol for SpyTag-SpyCatcher system verification

We tested our improved part BBa_K2684006 and the previous part BBa_K1583000 with the same protein expression system settings (E. coli BL21, pET28a vector, and the same induction and expression protocol).

The decrease of florescence of the supernatant indicates the immobilization of sfGFP-SpyCatcher, and there’s a significant difference between the two groups.

III. Verification - Using SpyCatcher-CotA

The immobilization of SpyCatcher-CotA fusion protein to the CsgA-SpyTag biofilm was tested, and a commercial laccase activity assay kits was used to measure the laccase activity.

Protocol for Biofilm x Laccase

The difference between our improved part and the previous part is significant. Thus, we proved that SpyCatcher-CotA can be attached to the biofilm containing functional peptide SpyTag.



IV. Conclusion

To conclude, we introduce a new ability to the previous CsgA part, which is capturing all fusion protein with SpyCatcher domain, by fusing SpyTag peptide to CsgA coding sequence. We verified the protein immobilization function of our improved part using sfGFP-SpyCatcher and SpyCatcher-CotA, and proved that SpyCatcher fusion protein is still functional after fixed to the biofilm.

Reference:

“Uranium Reaper.” Uranium Reaper - Team: Peking, 2016.igem.org/Team:Peking.