Experiments


General Experiments

Competent cell preparation

  1. 1 ml of an overnight E. coli culture was transferred to 100 ml of LB media and incubated at 37 °C and 200 rpm until OD = 0.4 - 0.6.
  2. The culture was poured into 50 ml falcon tubes, cooled on ice for 30 minutes and centrifuged at 4 °C and 4000 rpm for 5 minutes.
  3. The media was discarded, and the pellet was resuspended in 25 ml 0.1 M of ice cold MgCl2.
  4. The cells were cooled on ice for 30 minutes and spun down at 4 °C 4000 rpm for 5 minutes.
  5. The supernatant was discarded, the pellet was resuspended in 25 ml 0.1 M of ice cold CaCl2.
  6. The cells were cooled on ice for 30 minutes and spun down at 4 °C at 4000 rpm for 5 minutes.
  7. The supernatant was discarded, and the pellet was resuspended in 1.25 ml ice cold CaCl2/glycerol solution (1.7 mL 0.1 M CaCl2, 0.3 ml 100 % glycerol).
  8. The suspension was divided into 100 µl aliquots that were flash frozen in liquid nitrogen and stored at -80 °C.

Minipreps

  1. An overnight culture was grown and centrifuged at 4000 rpm for 5 minutes.
  2. The media was removed, and the pellet resuspended in 250 µl P1 buffer.
  3. 250 µl of P2 buffer were added, and the mixture was mixed well.
  4. 350 µl of N3 buffer were added, and the mixture was mixed well.
  5. The tube was centrifuged at 13000 rpm for 10 minutes, 800 µl of the supernatant were added to the QIAprep spin column.
  6. The column was centrifuged at 13000 rpm for 1 minute.
  7. 0.75 ml PE buffer were added to the column.
  8. The tube was centrifuged at 13000 rpm for 1 minute.
  9. The column was transferred to a new Eppendorf tube, 50 µl of EB buffer were added to the column.
  10. DNA was eluted after centrifuging 13000 rpm for 1 minute.

PCR cleanup

  1. 6X PB buffer was added to the PCR mixture and moved to the QIAquick spin column.
  2. The column was centrifuged at 13000 rpm for 1 minute, the flow through was discarded.
  3. 0.75 ml PE buffer were added to the column.
  4. The column was centrifuged again at 13000 rpm for 1 minute, the flow through was discarded.
  5. The column was centrifuged at 13000 rpm for 1 minute to remove any residual buffer.
  6. The column was transferred to a new Eppendorf tube and 30 µl of EB buffer was added.
  7. Purified DNA was eluted by centrifuging the column at 13000 rpm for 1 minute.

Transformations

  1. 10 µl of plasmid DNA or reaction mixture were added to 100 µl of competent E. coli cells and left on ice for 30 minutes
  2. The cells were heat shocked at 42 °C for 30 seconds, then returned to ice for 30 seconds.
  3. 1 ml of SOC media was added.
  4. The cells were incubated at 37 °C and 200 rpm for 45 minutes.
  5. The tube was spun down at 13000 rpm for 1 minute, the media was removed
  6. Bacterial pellet was resuspended in 100 µl of SOC, then plated.

Lysate preparation

  1. Overnight cultures of the transformed bacteria were grown at 37 °C
  2. The cultures were transferred to a 10 °C and 200 rpm shaker for 2 hours
  3. The cultures were spun down at 4000 rpm for 10 minutes
  4. The media was removed, and the pellets were resuspended in
    1. For PETases: PBS supplemented with 1mM EDTA and Roche’s cOmplete™ EDTA-free Protease Inhibitor Cocktail, 1 ml of PBS for 10 ml of culture pelleted.
    2. For other lysates: 0.4 M Tris-HCl (pH = 7.5), 150 mM NaCl, 1 mM DTT, 1 mM EDTA, EDTA-free protease inhibitor tablet (1 tablet per 50 ml) and 0.1% Triton.
  5. The resuspended cells were sonicated at 55% amplitude with 5 seconds of sonication and 10 seconds of rest time for 10 minutes.
  6. The tubes were spun down at 4000 rpm at 4 °C for 10 minutes to remove cell debris, the supernatant was kept on ice for immediate use or aliquoted and frozen at -80 °C for storage.

SDS-PAGE

  1. The tank for SDS-PAGE was set up, the inner chamber and half of the outer chamber were filled with 1X running buffer.
  2. 8 µl of sample lysate was mixed with 10 µl of 2X Laemmli sample buffer and 2 µl of β-mercaptoethanol under a fume hood.
  3. The mixture was boiled at 98 °C for 5 minutes, then spun down and either frozen at -20 °C or loaded onto a precast 4-15% Mini-PROTEAN TGX SDS-PAGE gel together with a protein standard.
  4. The power supply unit was connected to the electrodes and run at 180V until desired separation had been achieved.
  5. The gel was removed from the tank, carefully broken out of the plastic mould and washed with water.
  6. Water was poured off and enough Coomassie Blue to cover the entirety of the gel was added.
  7. The gel was incubated while shaking at an orbital shaker at ≈35 rpm until bands were clearly visible.

Restriction digests

  1. For a typical 20 µl reaction using EcoRI, the reaction was set up as follows:
    Reagent  Volume or mass added 
    DNA  1 µg 
    10X reaction buffer  2 µl 
    EcoRI  1 µl 
    H2O  Up 20 µl

  2. The mixture was incubated at 37 °C for 1 hour.
  3. The completed reaction was run on an agarose gel to visualise the products.

Colony PCR

  1. Colonies of interest were picked and resuspended in 50 µl of water.
  2. A reaction mixture was set up as below:
    Reagent  Volume added 
    Forward and reverse primers (10 µM)  0.25 µl 
    dNTP mix (10 mM)   0.5 µl 
    5X GoTaq reaction buffer  5 µl 
    GoTaq polymerase  0.125 µl 
    H2O  Up to 25 µl

    Forward primer: AGGGCGGCGGATTTGTCC; Reverse primer: GCGGCAACCGAGCGTTC


  3. The reaction mixture was added to a tube with 10 µl of the cell suspension.
  4. PCR was performed as follows:
    Temperature (°C)  Length  Number of cycles 
    95  0:03:00 1 
    95  0:00:15 30 
    55  0:00:15
    72  0:00:30
    72  0:05:00 1

  5. The completed mixture was run on an agarose gel to visualise whether the desired insert is present.

Error-prone PCR

  1. Dilute primers down to 10 uM
  2. Dilute DNA down to 1 ng/uL
  3. Set up the reaction using the table below
    PCR standard buffer  dNTP mix  55 mM MgCl2  0.01 mM MnCl2  FWD Primer  REV Primer  Taq Polymerase  Plasmid DNA  H2O  Total 
    5 uL  5 uL  5 uL  0.5 uL  1.5 uL  1.5 uL  0.5 uL  ~ 5 ng   to 50 uL  50 uL 

    dNTP concentrations were varied for each base (3.5 mM dATP, 4 mM dCTP, 6mM dGTP, 13.5 mM dTTP) to promote mutations
  4. Then run PCR using the cycling information as follows:
    1 cycle at 95°C for 60s,
    25 cycles at 95°C for 30s,
    1 cycle at 72°C for 45s,
    1 cycle at 68°C for 90s,
    elongation at 68°C for 5 mins then held at 4°C.
  5. Mutant MT plasmids were then purified and kept at -18°C.

Para-Nitrophenol butyrate (pNPB) Assay

  1. Prepare 50mM pNPB stock solution and reaction buffer (45 mM Na2HPO4-HCl (pH 7.0), 90 mM NaCl, and 10% (v/v) DMSO)
  2. The final reaction system contained protein samples (100-300 µg/ml), 2mM pNPB, and the reaction buffer.
  3. The reaction system was incubated for 30 minutes at 250 rpm 37°C for all PETase related constructs

JUMP Assembly

Level 0 Assembly

  1. The 20 µl reaction mixture was made up as follows:
    Reagent  Final concentration 
    P/R/N/O/C/T DNA part  1 nM 
    pJUMP-19 plasmid backbone  1 nM 
    10X T4 ligase buffer  1X 
    T4 ligase  1 U/20 µl 
    BsaI  10 U/20 µl 
    H2O  Up to 20 µl 

  2. The mixture was incubated in a thermocycler as outlined below:
    Temperature (°C)  Length  Number of cycles 
    37  0:15:00 1 
    37  0:03:00 30 
    16  0:03:00
    55  0:15:00 1 
    80  0:05:00 1

  3. The completed reaction mixture was subsequently used to transform the cells.

Level 1 Assembly

  1. The 20 µl reaction mixture was made up as follows:
    Reagent  Final concentration 
    P/R/N/O/C/T DNA part  1 nM 
    pJUMP-19 plasmid backbone  1 nM 
    10X T4 ligase buffer  1X 
    T4 ligase  1 U/20 µl 
    BsmBI  10 U/20 µl 
    H2O  Up to 20 µl

  2. The mixture was incubated in a thermocycler as outlined below:
    Temperature (°C)  Length  Number of cycles 
    42  0:15:00 1 
    42  0:03:00 30 
    16  0:03:00
    55  0:15:00 1 
    80  0:05:00 1

  3. The completed reaction mixture was subsequently used to transform the cells.

In vitro Transcription

Biosensor PCR

The original biosensor DNA was ordered from Twist Biosciences and resuspended in TE buffer. A PCR reaction was setup as follows:

Reagent  Final concentration 
DNA  50 pg/µl 
dNTP mix  2.5 mM 
5X Q5 reaction buffer  1X 
Forward and reverse primers  1.25 mM 
Q5 DNA polymerase  1.75 U / 20 µl 
DMSO  0.25% 
H2O  Up to 20 µl

The PCR reaction was then run as shown in the table below:

Temperature (°C)  Length  Number of cycles 
95  0:05:00 1 
95  0:00:30 30 
67  0:00:30
72  0:00:30
72  0:05:00 1

DNA transcription

The transcription of biosensors and the binding of the RNA aptamer to the fluorophore were tested using the reaction outlined below:

Reagent  Final concentration 
DNA  25 ng/µl 
DFHBI  0.1 µM 
10X T7 reaction buffer  1X 
dNTPs  1 mM 
T7 RNA polymerase  40 U / 20 µl 
H2O  Up to 20 µl

The reaction was allowed to proceed in the dark for 1 hour, the tube was then viewed under a blue lightbox to observe fluorescence and loaded onto an agarose gel to visualise any RNA production.


Protein Immobilization

Silica Immobilisation

  1. The lysates were diluted with 8 pH Tris- HCl to 500 µM.
  2. 20 mg of Celite were added to a tube and resuspended in 1 ml of the diluted lysate.
  3. The mixture was incubated at 4 °C and 200 rpm for 30 minutes.
  4. The tube was centrifuged at 10000 rpm for 10 minutes, the supernatant was discarded.
  5. The beads were resuspended in 8 pH Tris-HCl, the mixture was centrifuged at 10000 rpm for 10 minutes and the supernatant was removed.
  6. Step 5 was repeated twice.
  7. The beads were resuspended in 1 ml PETase buffer (45 mM Na2HPO4, 90 NaCl and 10% (v/v) DMSO).

Silica-Binding Assay for Characterising Silica Affinity Tags

  1. Dilute lysates to the protein concentration of 1 mg/ml using 0.4 M Tris-HCl (pH = 7.5)
  2. Add 20 mg of Celite545 into an eppendorf tube and resuspend it in 1 ml of the lysate
  3. Incubate the mixtures in a rotary mixer at 4°C for 1 hour.
  4. Centrifuge at 10000 rpm for 10 min.
  5. Move the supernatants to new tubes.
  6. Wash with Tris-HCl (pH=7.5).
  7. Assay the fluorescence of the initial (diluted) lysates and final supernatants, using the negative (Tris-HCl) control as the blank.

Hydrogel production

  1. 3% carboxymethyl cellulose solution was prepared by dissolving 30 g of CMC in 750 ml of DI water, stirring at room temperature until solution is even and adding 250 ml of DI water.
  2. 20 ml of the stock solution were transferred into a new container and 3 g of citric acid were added while stirring to make a 15% w/v citric acid/CMC solution.
  3. The solution was poured into casts and incubated at 40 °C for 24 hours to remove water.
  4. After dehydration, the temperature was increased to 80 °C and the mixture was incubated for a further 24 hours to facilitate crosslinking.
  5. The hydrogel was dehydrated at 40°C for 24 hours for storage.

CBD Fusion Protein CMC-Binding Assay

  1. Measure initial protein concentration based on Bradford assay.
  2. Dilute the lysates to 1 mg/ml by adding Tris-HCl (pH = 7.5)
  3. Cut out 20-30 mg of the hydrogel and transfer it to a Falcon tube
  4. Add 1 ml of lysate into the tube.
  5. Incubate the mixtures in a shaker at 4°C for 1 hour.
  6. Transfer the supernatants into new tubes.
  7. Assay the fluorescence of the samples, using Tris-HCl (pH = 7.5) as the blank.

Solubility test

  1. From an induced cell culture take 20 ml;
  2. Centrifuge cells at 4000 rpm for 5 min;
  3. Discard supernatant and freeze pellet at -20ºC for at least 30 min; (if pellet already frozen start from here!)
  4. Ressuspend in 2 ml of 1x PBS;
  5. Take a 100 µl sample at this point (sample 1);
  6. Sonicate in ice (5s pulses and 10s rest pulses);
  7. Centrifuge at 4000 rpm for 5 minutes;
  8. Take supernatant to a new tube; (sample 2)
  9. Resuspend pellet in 2 ml of 1x PBS; (sample 3)
  10. To 100 µl of each sample add 100 µl of 2x sample buffer and 10 µl of 2-mercaptoethanol;
  11. Run 20 µl in a 10% SDS PAGE gel.

ICP-MS Sample preparation

  1. The CBD-MT and lacZ lysates were thawed out on ice and diluted with 7.5 pH Tris-HCl to a protein concentration of 1 mg/ml.
  2. 1 ml of a diluted lysate was added to each hydrogel as shown in the following table.
    Hydrogel number Lysate added Metal added
    1 lacZ None
    2 lacZ None
    3 lacZ Zn2+ 
    4 lacZ Zn2+ 
    5 lacZ Zn2+ 
    6 lacZ Ni2+ 
    7 lacZ Ni2+ 
    8 lacZ Ni2+ 
    9 CBD-MT None
    10 CBD-MT None
    11 CBD-MT Zn2+ 
    12 CBD-MT Zn2+ 
    13 CBD-MT Zn2+ 
    14 CBD-MT Ni2+ 
    15 CBD-MT Ni2+ 
    16 CBD-MT Ni2+

  3. The hydrogels were incubated at a shaker for 1 hour at 4 °C and 150 rpm.
  4. The supernatant was discarded, and the hydrogels were washed with 7.5 pH Tris-HCl thrice.
  5. 1 ml of 7.5 pH Tris-HCl with either 100 µM Ni2+, 100 µM Zn2+, or containing no metal, was added to the hydrogel as shown in table 1.
  6. The hydrogels were incubated at a shaker for 1 hour at 37 °C and 200 rpm.
  7. 1 ml of supernatant from each tube was transferred into an Eppendorf tube for analysis using ICP-MS.


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