I. SpyTag-SpyCatcher System Verification

Make sure both preparation 1 and 2 are completed at the same time before the measurement.

Preparation 1:

1. Transfer Ptac Promoter - csgA – SpyTag in cell ΔMG1655 (Experiment Group)
2. Transfer Ptac Promoter – csgA in cell ΔMG1655 (Control Group)
3. incubate overnight
4. Dilute as 1:100 using LB media (both groups)
5. Incubate for 3 hours till OD≈0.6 (both groups)
6. Take 1.5ml of OD≈0.6 and add to 96 well plate (both groups)
7. Add IPTG, final concentration 0.1mM/L (both groups)
8. Induct in plate at 25℃ for 48h (both groups)
9. Centrifuge after induction and remove the supernatant (both groups)
10. Weigh the cells (both groups)

ΔMG1655 is MG1655 wild type with gene csgA knock out from its genome.

Preparation 2:

1. Transfer Ptac promoter - SpyCatcher – GFP (BBa_K2684004) into cell incubate overnight
2. Dilute as 1:100 with LB media
3. Incubate for 3 hours till OD≈0.6
4. Add IPTG, final concentration 0.1mM/L
5. Induct at 25 ℃ 220 rpm for 20 hours
6. Centrifuge and weigh cells
7. Add 5ml TBS buffer and ultrasonic to create reaction stock

Measurement:

1. Dilute reaction stock as 1:100 with PBS buffer
2. Measure florescence of the reaction stock after dilution
3. Add 1ml reaction stock after dilution to centrifuged cells from preparation 1 (to both group)
4. React for 1 hour
5. Centrifuge
6. Measure florescence of the supernatant
7. How much florescence the reaction stock lost could indicate how much the palette gains. In that logic, how much sfGFP-SpyCatcher protein the palette gains could be indicated by the difference between the reaction stock after dilution (step2) and the supernatant after reaction (step6).

II. Laccase Activity Assay, Scale of Protein

III. Laccase Activity Measuring Kit

IV. Biofilm x Laccase