Team:SHSBNU China/Protocol

Section Sample

Protocol

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

Subjects:

  • Extracted liquid: 1 glass of 100mL liquid at 4 degrees Celsius.
  • Reagent solution 1: 1 glass of 15mL liquid at 4 degrees Celsius.
  • Working fluid: 2 glasses of powder at 4 degrees Celsius, kept at dark place. Add into 7.5mL of reagent solution 1 before use.

Description:

Laccase(CE.10.3.2) is a copper-containing polyphenol oxidase; it is an enzyme that can be found in many plats, fungi, and microorganisms. It has a strong redox potential, and is widely applied in various real-life settings such as biobleaching of paper pulp, decomposition of wastes, bioinstrumentation and decomposition of lignocelluloses.

Laccase decomposes substrate ABTS, producing ABTS free radical, absorption coefficient is much larger at 420nm compared to substrate ABTS, measure rate of increase of ABTS free radical in order to calculate laccase activity.


Instrument and reagent:

balance, low temperature centrifuge, UV-visible spectrophotometer, microplate reader, raman quartz cuvette/96 pore plate, and a laboratory water bath at constant temperature.


Procedure of extracting enzyme liquid:

  1. Tissue: lower the temperature using mixture of ice and water, and homogenate the mixture of tissue and the extracted liquid (mass of tissue : mass of liquid as 1 : 5-10; 0.1g tissue and 1mL extracted liquid suggested). Centrifuge at 4 degrees Celsius for 10 minutes, 10000g, draw the supernatant, and place it on ice for further measurement.
  2. Cell: lower the temperature of the mixture of cells and the extracted liquid using mixture of ice and water, then add ultrasonication to destroy the cells of the mixture for 3 minutes, 300w power (number of 104 cells : volume of the extracted liquid as 500-1000 : 1mL; 5 million cells and 1mL extracted liquid suggested)
  3. Cell nutrient solution: measure directly. Operation form shown below.
Control Tube Experimental Tube
Sample (uL) 25
Boiling sample (uL) 25
Working fluid (uL) 150 150
60 degrees Celsius, 3 minutes, measure light absorption value A on raman quartz cuvette/96 pore plate at 420nm, ΔA=A experimental group – A control group

Formula to calculate laccase activity using sample mass:

Definition of laccase activity: 1 laccase activity is the amount of enzyme needed for every sample (per gram) to oxidize 1nmol substrate ABTS per minute.

laccase activity (nmol/min/g) = ΔA /ε(ABTS mmolar extinction coefficient) / d * V (total volume of reaction) / V (volume of sample in the reaction, 0.025mL) / W (sample mass, g) * V (extracted liquid added, 1mL) / T (reaction time, 3 minutes) = 130 *ΔA / W

IV. Protocol for Amyloid Protein Verification

Media: LB

  1. Transfer
    • plasmid pET28a Neg Control group
    • Ptac promoter-CsgA-Spytag Experiment group
  2. Pick MG1655 wild type Pos Control group
  3. Incubate
  4. Streaked all three groups on pigmented plates
    • These plates were supplemented with 0.5mM of IPTG, 25 ugml-1 of Congo Red and 5 ugml-1 of Brilliant Blue G250.
  5. The plates were then incubated for 48h at 25 degrees Celsius

V. Biofilm x Laccase

Protocol of Biofilm

  1. Transfer cells
    • Host: ΔMG1655 (MG1655 without csgA on genome)
    • Group A: Pet28a plasmid
    • Group B: Ptac Promotwe – csgA - Spytag
    • Host: BL21 - DE3
    • Group C: T7 promoter - cotA
    • Group D: T7 promoter - SpyCatcher – cotA
    • 2. Incubate Overnight
    • 3. Dillute as 1:100 using LB media
    • 4. Incubate for 3 hours till OD≈0.6
    • 5. Add IPTG, final concentration 0.1 mM/L
    • 6. Induct for 3 hours at 30℃, 180 rpm
    • 7. Leave at rest for 20 hours
    • 8. Centrifuge
    • 9. Transfer the supernatant of group C and D to other containers
    • 10. Weigh the palette of 4 groups
    • 11. Add 5ml TBS buffer in C and D and ultrasonic to create reaction stock C and D
    • 12. Re-suspend group A and B using2 ml of TBS buffer
    • 13. Mix
      • 1ml of A + 1ml of reaction stock of C - Group1
      • 1ml of A + 1ml of reaction stock of D - Grouo2
      • 1ml of B + 1ml of reaction stock of C - Group3
      • 1ml of B + 1ml of reaction stock of D - Group4
    • 14. React at 37℃ for 1hour
    • 15. Centrifuge
    • 16. Remove supernatant
    • 17. Re-suspend group A and B using 1 ml of PBS buffer

Measurement:

  1. Step 10 above.
  2. Measure laccase activity using commercial kit
    1. Group1 of step13
    2. Group2 of step13
    3. Group3 of step13
    4. Group4 of step13
    5. Supernatant of C
    6. Reaction stock of C
    7. Supernatant of D
    8. Reaction stock of D