Difference between revisions of "Team:SHSBNU China/Improve"
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#ID">Improvement Details</a> | <a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#ID">Improvement Details</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="trd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#b">CotA Improvement</a> | ||
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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: | 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: | ||
Revision as of 20:06, 17 October 2018
Improve
Improve
Overview
Previously, there was only CsgA part uploaded in iGEM parts website. Our Team improved part Part BBa_K1583000 by adding a SpyTag sequence. It fused to gene csgA, enabling CotA laccase to be fixed onto the biofilm to form a covalent bond--SpyTag-SpyCatcher.
Improvement Details
CsgA Improvement
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 csgA on the plasmid of pET28a was transferred into ΔMG1655 as control group. Gene csgA – spytag 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 csgA nor SpyTag in neither genome nor plasmid; the ones in Group 1 had csgA in the genome; the ones in Group 2 had sfGFP in the plasmid; the ones in Group 3 had csgA-SpyTag in the genome and the plasmid. The treatment of the experiment is followed by the procedure below:
The liquid after centrifugation was extracted and measured fluorescence.
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.
Part BBa_K2684000 was improved from previous part BBa_K1336002. 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:
The improvement is valid since it was normally produced.