Team:SHSBNU China/Experiments
Experiment
I. Outline
Our system includes two main parts one is CotA laccase and the other is biofilm containing SpyTag. So our experiments were mainly divided in three periods, verifying both parts individually and verifying Biofilm x Laccase system as a whole.
The first section was to construct our biofilm containing SpyTag. We fused SpyTag to the CsgA, which is the major component of our biofilm. We successfully verified that protein fused with spycatcher domain could be fixed on our biofilm.
The we isolated CotA gene of B. subtilis WT168 and successfully expressed this laccase in E. coli cell. We confirmed its oxidation function. Furthermore, we attached CotA coding sequence with several signal peptides and tested the efficiency of secretion.
The final section is mainly focused on combination of the two parts above. 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.
III. Period 2 - CotA - SpyCatcher
Gene cotA from B. substiles could translate as laccase protein (BBa_K2684000). The sequence was fused on to pET28a plasmid.
We then added the SpyCatcher sequence after cotA gene so that we can produce laccase protein with spycatcher (BBa_K2684005).
We also figured a way to secret our CotA protein out of the host cell. Signal peptides are small parts of protein and their function is to lead whatever behind it out of the cell. A signal peptide sequence was also added ahead of cotA sequence. Three signal peptides was tested: Pelb, PhoA, OmpA (BBa_K2684001, BBa_K2684002, BBa_K2684003).
ABTS is a substance that is wildly used in commercial laccase activity assay kits. ABTS is in a clear solution and when it reacts with laccase, it would turn to a bullish-green color that has an absorbance peak at 420nm. We used laccase activity assay kit from Solarbio, type BC1635 which functions based on the previous mentioned method. We set a negative control group by transferring pET28a plasmid in to BL21 – DE3 strain. We set 4 experiment groups by transferring cotA, OmpA - cotA, PhoA - cotA, PelB – cotA gene into BL21 – DE3. We measured the laccase activity of all 5 groups mentioned above to check if our laccase is functional.
Link Protocol for laccase activity assay, scale of protein
Link Protocol for Laccase activity measuring kit
IV. Period 3 - Biofilm x Laccase
The final period is to assay the laccase activity of Biofilm x Laccase system as a whole. Four group of experiments was conducted.
| Group name (in protocol, step 13) | Biofilm + SypTag | Enzyme |
| 1 | No | CotA |
| 2 | No | CotA - SpyCatcher |
| 3 | Yes | CotA |
| 4 | Yes | CotA - SpyCatcher |
The method of centrifuging and removing the supernatant was to remove uncombined enzyme after the reaction of SpyTag and SpyCatcher (Step 14).
Link Protocol for Biofilm x laccase
Link Protocol for Laccase activity measuring kit
