Difference between revisions of "Team:SHSBNU China/Experiments"
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Gene <i>csgA</i> found in the genome of MG1655 wild type is capable of forming biofilm. Using CRISPR, we knocked out gene <i>csgA</i> on MG1655’s genome creating ΔMG1655 strain. The cell ΔMG1655 would then be used as chassis cell. Gene <i>csgA</i> was fused into plasmid pET28a. A <i>Spytag</i> sequence was then fused after <i>csgA</i> gene, creating <i>csgA-spycatcher</i> (BBa_K2684006). | Gene <i>csgA</i> found in the genome of MG1655 wild type is capable of forming biofilm. Using CRISPR, we knocked out gene <i>csgA</i> on MG1655’s genome creating ΔMG1655 strain. The cell ΔMG1655 would then be used as chassis cell. Gene <i>csgA</i> was fused into plasmid pET28a. A <i>Spytag</i> sequence was then fused after <i>csgA</i> gene, creating <i>csgA-spycatcher</i> (BBa_K2684006). | ||
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Using sfGFP – spycatcher protein, the combing function of Spytag and spycatcher system on the biofilm was tested. Gene <i>csgA</i> on the plasmid of pET28a was transferred in to ΔMG1655 as control group. Gene <i>csgA – Spytag</i> on the plasmid of pET28a was transferred in to ΔMG1655 as experiment. To verify the function of Spytag on <i>csgA</i>, the experiment was design to compare the combing rate of sf-GFP – spycatcher protein with cells that have csgA – SpyTag (Experiment) or csgA (Control). | Using sfGFP – spycatcher protein, the combing function of Spytag and spycatcher system on the biofilm was tested. Gene <i>csgA</i> on the plasmid of pET28a was transferred in to ΔMG1655 as control group. Gene <i>csgA – Spytag</i> on the plasmid of pET28a was transferred in to ΔMG1655 as experiment. To verify the function of Spytag on <i>csgA</i>, the experiment was design to compare the combing rate of sf-GFP – spycatcher protein with cells that have csgA – SpyTag (Experiment) or csgA (Control). | ||
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Link: Protocol for <a>SpyTag-SpyCatcher</a> system verification | Link: Protocol for <a>SpyTag-SpyCatcher</a> system verification | ||
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<img class="pictures" id = "21002" src="https://static.igem.org/mediawiki/2018/5/57/T--SHSBNU_China--21002.jpg"/> | <img class="pictures" id = "21002" src="https://static.igem.org/mediawiki/2018/5/57/T--SHSBNU_China--21002.jpg"/> | ||
Revision as of 00:05, 17 October 2018
Experiment
I. Outline
Our system includes two main components as mentioned previously. So our experiments were mainly divided in three periods, verifying both parts individually and verifying Biofilm x Laccase system as a whole.
The first period was to construct our biofilm + Spytag, which would be used for combination later on. This period included creating part BBA_K2684006, csgA – SpyTag and BBA_K2684004, sfGFP – SpyCatcher. As well as improving a previously part, BBa_K1583000, csgA. The biofilm + SpyTag ’s ability of combining protein domains + SpyCatcher was measured and our biofilm with SpyTag system tend to be successful.
The second period is focused to construct and measure the laccase activity of our parts: BBA_K2684000, CotA, BBA_K2684001, Pelb - cotA, BBA_K2684002, PhoA - cotA, BBA_K2684003, OmpA - cotA, BBA_K2684005, cotA - SpyCatcher. The laccase activity of the 5 parts were measured.
The final period is mainly focused on combination of period 1 and 2 which means constructing our Biofilm x Laccase system. Experiments were conducted to measure the laccase activity of the total Biofilm x Laccase system. A significant difference was confirmed between the experiment group and the control group. Thus, our system is successful.
II. Period 1 - CsgA - SpyTag
Gene csgA found in the genome of MG1655 wild type is capable of forming biofilm. Using CRISPR, we knocked out gene csgA on MG1655’s genome creating ΔMG1655 strain. The cell ΔMG1655 would then be used as chassis cell. Gene csgA was fused into plasmid pET28a. A Spytag sequence was then fused after csgA gene, creating csgA-spycatcher (BBa_K2684006).
Reaction stock leftover in experiment
Using sfGFP – spycatcher protein, the combing function of Spytag and spycatcher system on the biofilm was tested. Gene csgA on the plasmid of pET28a was transferred in to ΔMG1655 as control group. Gene csgA – Spytag on the plasmid of pET28a was transferred in to ΔMG1655 as experiment. To verify the function of Spytag on csgA, the experiment was design to compare the combing rate of sf-GFP – spycatcher protein with cells that have csgA – SpyTag (Experiment) or csgA (Control).
Link: Protocol for SpyTag-SpyCatcher system verification
As can be seen from the result,
Thus we can confirm our csgA – SpyTag system is functional.