Team:SHSBNU China/Protocal

Section Sample

Protocal

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

Prepare:

Negative Control Group: pET28a empty plasmid

Experimental Group：

  1. T7 promoter - LacO - cotA
  2. T7 promoter - LacO – OmpA - cotA
  3. T7 promoter - LacO – PhoA - cotA
  4. T7 promoter - LacO – PelB – cotA

Bacterial Strain: e. coli BL21 – DE3

Medium: LB


Procedure:

  1. Incubate experiment group 1,2,3,4 and control group overnight
  2. Draw 10uL overnight broth,
  3. Dilute as 1:100 using 10mL LB medium
  4. Transfer to 100mL shake flask at 30 degrees Celsius
  5. Incubate at 30 ℃, 180 rpm for 3 hours, till OD≈0.6
  6. Add IPTG (final concentration as 0.1mM/L)
  7. Add CuSO4(final concentration as 0.1mM/L) at 25 degrees Celsius, induct at 180rev/min for 4 hours
  8. Stop shaking; Incubate for another 20 hours at 25 degrees Celsius, let bacteria grow under micro-aerobic condition
  9. Draw some sample from all groups and dilute it as 1:5, measure OD600, control OD600 at the same level.
  10. Centrifuge all groups.
  11. Add 5mL PBS buffer to the bacteria in order to let it suspend
  12. Ultrasonic
  13. Draw 25uL from all groups respectively, then continue the experiment by following the instruction on laccase activity assay kit

III. Laccase Activity Measuring Kit

IV. Biofilm x Laccase