▼ EXPERIMENT PROTOCOL ▼

Overview

This tab documents the experiments conducted, materials used, and compiled protocols used by the SPC iGEM team in the creation of the final biological enzymes.

The experimental workflow includes:

  1. Transformation of E. coli (DH5α and BL21) with plasmids
  2. Culturing the transformed E. coli in medium
  3. IPTG induction of samples
  4. His-tag purification for quantitative and qualitative analysis of modified PETases expression
  5. SDS-PAGE analysis
  6. PET degradation assays to measure modified PETases functionality
  7. Plasmid detection via gel electrophoresis

1. Transformation

1.1. Overview

Introduce plasmids into competent cells in order to replicate the plasmid in E. coli.

1.2. Materials

1.2.1. Preparation of LB Agar Plate

  • Kanamycin stock (50mg/MilliQ 1mL)
  • LB agar medium (LB agar powder 9.9g, MilliQ 300mL)

1.2.2. Materials transferred to LB agar plates

  • Competent cells
  • TE buffer (20x)
  • Plasmid DNA
  • S.O.C. medium

1.3. Protocol

1.3.1. Preparation of LB agar plates

Add 9.9g of LB agar powder to MilliQ such that the final volume of the solution is 300mL
Add 0.3mL of 50mg/mL kanamycin stock to 300mL LB agar solution, the final concentration of kanamycin should be 50 μg/mL
Pour 12.5mL of the LB/kanamycin solution into 8 petri dishes and allow to set

1.3.2. Transformation

Dilute the TE buffer with MilliQ from 20x to 1x
Dispense 50 µL of competent cell into 1.5 mL microcentrifuge tubes and thaw it on ice
Add 2~5 µL of plasmid DNA into the microcentrifuge tube of competent cells and mix them well by gently shaking the tubes
Incubate the cells on ice for 10 minutes
Heat-shock the cells for 30 seconds in a 42°C water bath
Add 125 µL of room-temperature S.O.C. Medium and shake the tube at 225 rpm for 1 hour at 37°C
Resuspend the cells in TE buffer
Streak 100 µL of cells onto the prepared LB agar plates
Label and incubate the plates via spinning incubator overnight at 37°C, 225rpm
Count the number of colony-forming units (CFUs) for each petri dish
Streak the plates every 1-2 weeks so as to preserve the transformed E. coli

2. Culture the Transformed Cells

2.1. Overview

Culture the transformed cells in YESCA medium with kanamycin

2.2. Materials

  • YESCA medium (10 g/L casamino acids, 1 g/L yeast extract), 100 mg/L kanamycin
  • Transformed cells

2.3. Protocol

Add 100μL of kanamycin solution per 40mL of YESCA solution
Pick colonies formed from the 4 prepared colonies of BL21 E. coli (HotPETase, CsgA-HotPETase, HotPETase-BaCBM2, and CsgA-HotPETase-BaCBM2) from the LB agar plates
Add 45mL of YESCA kanamycin solution to each test tube
Innoculate the colonies into corresponding test tubes with YESCA medium
Cultivate overnight at 37°C at 225rpm
Seal petri dishes containing transformed cells with laboratory parafilm and place in 4°C refrigerator

3. IPTG Induction

3.1. Overview

IPTG induction is the process to induce expression of the four types of modified PETases by adding IPTG to E. coli BL21, the samples of which were transformed prior to IPTG induction.

3.2. Materials

  • YESCA medium
  • 0.1M IPTG solution

3.3. Protocol

Dilute the colonies in YESCA medium so that the samples have an OD600 of 0.05
Inoculate the cells in fresh YESCA medium with the initial OD600 of 0.05 to a total volume of 25 mL and cultured about 3 hours at 30°C and 225 rpm
Measure OD600 using 1 mL of the culture solution
Confirm the OD600 to be 0.5 and divide the sample into three 15 mL portions. Add 96 µL of 100 mM IPTG solution to all of the portions
Add 96 µL of 100 mM IPTG solution to all of the portions
Incubate samples prepared in step 5 for 22 hours at 25°C, 225 rpm

4. His-spin and His-tag Purification

4.1. Overview

Expressed His-tagged proteins are purified and detected as the string of histidine residues binds to several types of immobilized metal ions.

4.2. Materials

  • His‑Affinity Gel
  • His‑Binding/Wash/Elution buffers
  • Zymo‑Spin P1 columns
  • Collection tubes

4.3. Protocol (His-spin)

Transfer 250 μl of His-Affinity Gel to the Zymo-Spin P1 column, then place it into a collection tube. Make sure the resin is fully resuspended by shaking/vortexing the bottle before transfer by way of pipette
Centrifuge for 5-10 seconds. Ensure that the His-affinity gel is fully drained. Some older centrifuge models may require a longer duration of centrifugation. However, do not centrifuge the gel for too long due to risk of overdrying
Add 150-300 μl of protein sample and resuspend the gel by shaking or tapping the column
Resuspend the gel for a few more times during a two-minute incubation period. It is imperative that the sample is allowed to interact with the gel for at least 2 minutes. For any sample volume >200μl, additional binding time may be needed to improve the yield of purified protein
Add 250 μl of His-Wash Buffer and resuspend the gel. Centrifuge for 5-10 seconds
Repeat step #5 once more. Discard the collection tube
Place the Zymo-Spin P1 column into a standard microcentrifuge tube
Add 150 μl of His-Elution Buffer to the column and resuspend the gel. Elution volumes can vary between 100-200μl. 150 μl of His-Elution Buffer elutes virtually all the column-bound protein. Smaller elution volumes are also possible and may yield more concentrated protein, but the elution efficiency may be compromised
Centrifuge 5-10 seconds to elute the purified protein

5. SDS-PAGE

5.1. Overview

SDS-PAGE is the process to confirm whether the modified PETases are expressed or not by determining a protein's size and assess its purity.

5.2. Materials

5.2.1. His-tag purification

  • Lysed cells
  • His-wash buffer
  • His-elution buffer

5.2.2. Preparation of protein sample

  • SDS running buffer (20x)
  • LDS sample buffer (blue)
  • Reducing agent Buffer
  • Appropriate protein samples
  • MilliQ

5.2.3. Gel electrophoresis

  • SDS running buffer
  • Protein ladder (20 μL per gel)
  • Prepared protein samples

5.2.4. Staining of gel

  • MilliQ
  • SimplyBlue SafeStain (20 mL per gel)

5.3. Protocol

5.3.1. Freeze thaw for cell lysis

Place the IPTG-induced solution under -80°C freezer for 20 minutes
Take the solution out of the freezer and submerge the sealed solution in 35°C water bath for 5 minutes
Repeat steps 1 & 2 for 6 times

5.3.2. His-tag purification in preparation for SDS-PAGE

Collect lysed cells for each type of DNA
Centrifuge at 12000xg for 5 minutes
Resuspend the gel in 150μl of each centrifuged sample
Incubate at 225rpm at room temperature for 2 minutes
Centrifuge the samples at 12000xg for 10 seconds
Add 250μl of His-wash buffer to the gel. Then resuspend the gel
Centrifuge the samples at 12000xg for 10 seconds
Repeat steps #6 and #7 once
Add 150μl of His-elution buffer to each column. Resuspend the gel
Centrifuge the samples for 10 seconds
Collect and label the eluates

5.3.3. Preparation of protein sample for SDS-PAGE

Dilute the SDS running buffer with MilliQ from 20x to 1x. Store the diluted buffer in a 4°C refrigerator
Make up a 50μl sample for each protein prepared in section 5.3.1. The sample should consist of 17.5μl protein sample, 12.5μl LDS sample buffer, 5μl reducing agent buffer, and 15μl MilliQ

5.3.4. Gel electrophoresis (SDS-PAGE)

Prepare the gel column
Fill the gel chamber with approximately 400mL 1x SDS running buffer
Place the prepared gel column into the electrophoresis chamber. Rinse the wells with 1x SDS running buffer
Load 20μl of protein ladder to the leftmost well. Load other protein samples in no particular order. Do not load more than one sample or ladder into one well
Connect the setup to a constant 150V supply for approximately 30 minutes

5.3.5. Staining of gel

Remove the gel columns from the chamber
Pry open the plastic casing and remove the gel
Rinse gel with MilliQ twice
Prepare a tray of 20mL SimplyBlue SafeStain solution
Submerge the gel into the solution
Swirl the gel constantly for 1 hour in the incubator at 35rpm, at room temperature

6. Test for PET Degradation

6.1. Overview

To determine the extent to which the modified PETases were successful in degrading PET under different temperatures, namely 25°C, 60°C, and 70°C. Pieces of PET plastic were used as samples of degradation. Mass change of the plastic and absorbance of the modified PETases samples were the two metrics used in assessing the results of degradation.

6.2. Materials

  • IPTG induced samples
  • PBS solution
  • PET pieces

6.3. Protocol

Centrifuge the expression-induced samples at 9000 xg, for 15 minutes
Further centrifuge all solutions at 15200rpm for 25 minutes
Remove the supernatant
Resuspend the modified PETases' pellet in pH 7.4 PBS solution
Calibrate the spectrophotometer with PBS solution. Conserve the PBS solution and calibrate once every day
Model the thermal conditions using an incubator or water bath at 60°C and 70°C respectively. Centrifuge the sample solutions at 13000 rpm for 5 minutes and record the absorbance of the supernatant and mass change of the dried PET plastic every 24 hours. Control samples of PBS solution with PET plastic are used for every thermal condition

7. Plasmid Detection

7.1. Overview

To prepare plasmid DNA from recombinant E. coli cultures at a small scale.

7.2. Materials

  • Resuspension solution
  • Lysis solution
  • Neutralization solution
  • Concentrated wash solution
  • RNase A (10 mg/mL)
  • Elution Buffer (10 mM TrisHCl, pH 8.5)
  • GeneJET™ Spin Columns
  • 96% ethanol
  • Transformed E. coli

7.3. Protocol

Add 170mL of 96% ethanol to 100mL of concentrated wash solution
Resuspend pelleted cells in 250 μL of Resuspension Solution. Transfer cell suspension to a microcentrifuge tube, then resuspend completely by vortexing or pipetting up and down until no cell clumps remain
Add 250 μL of Lysis Solution, then mix gently by inverting the tube 4–6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing chromosomal DNA
Transfer the GeneJET™ spin column into a fresh 1.5 mL microcentrifuge tube
To elute the plasmid DNA, add 50 μL of the Elution Buffer to the center of the spin column membrane
Incubate for 2 minutes at room temperature, then centrifuge ≥12,000 xg for 2 minutes. Discard the spin column
Store the purified plasmid DNA at -20°C
Mix 4 components together according to the following order: 24 μL of deionized water, 4 μL of FastDigest Green Buffer, 8 μL of DNA, and 4 μL of Fast Digest Enzyme
Incubate the reaction mixture at 37°C for 5 minutes
Inactivate the enzyme at 80°C for 5 minutes
Load aliquots of 15 μL of each DNA mixture into the gel
Connect the setup to a constant 220V supply for approximately 15-20 minutes