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. 

GWe appended peptide domain SpyTag to amyloid protein CsgA, the dominant component in E. coli biofilms [Fig 1.1]. The sequence of this fusion protein was constructed on pET28a plasmid and was promoted by a Ptac promoter. Since there is already a copy of CsgA gene on MG1655 wild type’s genome, we need to knock out this wildtype gene CsgA. Using CRISPR, gene CgsA successfully knocked out the gene on MG1655’s genome (ΔCsgA-MG1655 strain, done by Yiming Dong from Bluepha). The strain ΔCsgA-MG1655 would then be used as chassis cell in afterward experiments.

Verification of biofilm forming

Amyloid protein, like CsgA, could be colored by certain protein pigment. For instance, brilliant blue and Congo red. To verify the biofilm forming after IPTG induction, we compared the biofilm configuration between different experiment groups.

Protocol for amyloid protein verification

We discover [Fig1.2] that colonies of CsgA-SpyTag and the positive group show similar color and spreading pattern, while the colonies of the negative control group was colored to dark purple. Thus, our CsgA-SpyTag amyloid protein was successfully expressed.

We designed another experiments to measure the capture ability of CsgA–SpyTag [Fig 1.3]. We used sfGFP – SpyCatcher protein as the indicator [Fig 1.4]. Gene CsgA on the plasmid of pET28a was transferred in to ΔCsgA-MG1655 as control group. Gene CsgA – Spytag on the plasmid of pET28a was transferred in to ΔCsgA-MG1655 as experimental group. We used a sfGFP – SpyCatcher protein containing reaction stock, of which the fluorescence is measured, to react with the palette of both groups for 1 hour. Then, we centrifuged the reaction system and measure the fluorescence of the supernatant. The decrease of florescence indicated the amount of sfGFP – SpyCatcher protein the biofilm captured.

Protocol for SpyTag-SpyCatcher system verification

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 is significant. Thus we can confirm our CsgA-SpyTag system is functional.

After that, we test our biofilm’s ability to attach to surfaces for our hardware design. We induce ΔCsga-MG1655 with Ptac promoter-CsgA-SpyTag while PHA plastic beads were also soaked in the culture [Fig 1.6]. The PHA beads after 20 hours of induction was set as experiment group. Untreated, beads were set as control group.

We then use brilliant blue G250 to dye the both the experiment group and the control group. We allow 5 minute before washing off the extra dye with water for several time.

There is a visual difference [Fig 1.7] between the control and the experiment group, whit the experiment slightly more blue that the control group.

Thought having a naked-eye distinguishable difference group between the experiment and control group, the difference was limited. In other words, it means our biofilm’s ability to stick to untreated PHA plastic beads is limited. To stick our biofilm on those beads, we need certain modifications like carving drills on PHA plastic beads.

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

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