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TIMELINE & ACTIVITIES

Laboratory Timeline

CA cloning, induction & analysis

RBS-GFP (I13500) cloning, induction & analysis

CYP2E1 cloning, induction & analysis

HP timeline

Education & public engagement

Integrated human practice

EXPERIMENTAL PROTOCOL

Plasmid Extraction

Bacterial culture

For plasmid extraction, we normally cultured E. coli carrying plasmids in 6 ml LB broth supplemented with chloramphenicol (34 μg/ml) or ampicillin (100 μg/ml) at 37°C overnight.

Plasmid Extraction Procedure

In all of our experiments, we used a mini-prep kit (Presto™ Mini Plasmid Kit) developed by GENEAID BIOTECH LTD., Taiwan. And we followed the manufacturer's instructions for plasmid extraction. Below is the brief protocol provided by Geneaid Biotech Ltd., Taiwan. And you can also go to their website for more information and complete protocol.

PCR & Colony PCR

We always used a high fidelity DNA polymerase named KOD -Plus- provided by TOYOBO CO., LTD. We followed the manufacturer's instructions. Conditions and reagents were briefly shown below.

Reagents for PCR

Conditions of PCR

Colony PCR

We used a kit of Taq polymerase (JMR Holdings) and followed the manufacturer's instructions. Brief conditions were shown below.

Reagents for colony PCR

Conditions of colony PCR

DNA Gel Electrophoresis

We prepared 1% DNA agarose gels and run gel with 6X FluoroDye DNA Loading Dye (Green) and FluoroBand 1KB (0.25-10kb) Fluorescent DNA Ladder. The images were pictured with Omega Lum™ G Imaging System.

PCR Cleanup & Gel Elution

We used GenepHlow™ Gel/PCR Kit developed by GENEAID BIOTECH LTD., Taiwan and followed the manufacturer's instructions.

Restriction Enzyme

EcoRI, XbaI, SpeI, PstI were used in this study and purchased from New England Biolabs (NEB). We have three conditions as described below used for plasmid check and gene cloning, respectively.

E.coli DH5a Transformation

Competent cells

We used and purchased Elite Competent Cells (DH5α) from GENEAID BIOTECH LTD. We followed the provided protocol for transformation procedure and briefly described below.

Transformation

↓ Thaw one tube of competent cells on ice from -80°C refrigerator
↓ Add 5 μl of DNA into 50 μl of competent cells. Operate in a laminar flow and manipulate DNA and competent cells on ice.
↓ Vortex for 1 sec and stay on ice for 5 min
↓ Heat-shock the cells at 42°C for 1 min
↓ Incubate on ice for 5 min
↓ Recover with 500 μl of LB media and shaking in an incubator at 37°C for 1 hr. Operate in the laminar flow.
↓ Spread the cells onto a LB agar plate supplemented with 34 μg/ml of chloramphenicol or 100 μg/ml of ampicillin. Operate in the laminar flow.

Bacillus subtilis 168 Transformation

To transform Bacillus, we used the electroporation method created by Team UBonn_HBRS in iGEM 2016.

Making electro-competent cells

↓ Inoculate a liquid culture of Bacillus subtilis and let it grow overnight
↓ Vortex the culture gently and transfer 500 μl in 100 ml competency medium in the flask, make 3 repeats
↓ Grow the bacteria in flasks at 37°C, shaking at 200 rpm until reaching an OD650 between 0.5 and 0.7
↓ Add 1ml, 1.25ml and 1.5ml of 20% glycine solution to the culture media
↓ Keep shaking for 1 hr
↓ Cooldown the cells on ice for 15 mins
↓ Centrifuge at 8500 rpm for 10 min at 4°C to get bacterial pellets
↓ Pour off the supernatant and wash the cells three times with ice-cold washing buffer (20 ml, 10 ml, 5 ml, respectively). Centrifuge the cells down at 8500 rpm for 10 min at 4°C between each washing procedure and decant the supernatant
↓ Resuspend the cells in 1 ml ice-cold washing buffer
↓ Make aliquots with 120μl to one eppendorf (one vail is enough for two reactions in transformation)
↓ Immediately freeze in liquid nitrogen and store at -80°C

Electroporation transformation for Bacillus

Electroporation is a transformation method that relies on providing an electric current through the cell and creating holes or pores in the cell membrane. Through those pores, the plasmid will enter the cell before they are getting closed.
To do this transformation, the equipment and materials needed are listed below:

  1. Electrocompetent cells
  2. Competency medium
  3. Plasmid you want to transform
  4. Electroporator
  5. Electroporation cuvettes

Procedure:
↓ Mix electrocompetent cells with the plasmid and acquire a total volume of 60 μl with a final DNA concentration of 10 ng/μl
↓ Place the electroporation cuvettes with Bacillus cells and plasmids in competency medium on ice for 10 min (1 ml of competency medium & 1 electroporation cuvette per transformation)
↓ Pipette the cold the mixture of cell and DNA in the prechilled electroporation cuvette and tap the cuvette multiple times
↓ Electroporate at 2100 Volt and set a time constant from 3.0 to 5.5
↓ Flush the electroporated mixture out of the cuvette with 1 ml of competency medium
↓ Let the cells grow for 3 hr at 37°C, shaking at 300 rpm
↓ Pellet cells by centrifugation at 5 min and decant the supernatant
↓ Resuspend the pellet in 100 μl of the supernatant
↓ Plate on selective agar

Protein Induction Procedure

GFP

↓ culture Bacillus subtilis 168 carrying the plasmid of PliaI + RBS + GFP + terminator/pBS0E in LB supplemented with erythromycin (1 μg/ml) and lincomycin (25 μg/ml) O/N at 37°C, shaking at 170 rpm
↓ transfer 3 ml to 50 ml LB with antibiotics in 250 ml flask
↓ measure OD650
↓ shake at 200rpm,37°C until OD650 between 0.5~0.7
↓ add 30μg/ml of bacitracin for induction at 25°C, shaking at 100 rpm inthe incubator for 18.5 hr

CA

↓ culture Bacillus Subtilis 168 carrying the plasmid of PliaI + RBS + hCA + terminator/pBS0E in LB + antibiotics O/N at 37°C, shaking at 170 rpm, supplied with 60μM ZnSO4
↓ transfer 3 ml to 50 ml LB+Antibiotics in 250 ml flask with 60μM ZnSO4
↓ measure OD650
↓ shake at 200rpm,37°C until OD650 between 0.5~0.7
↓ add 400μM ZnSO4 and 50μl of 30μg/ml Bacitracin for induction at 25°C, shaking at 100 rpm for 18.5 hr

CYP2E1

↓ culture Bacillus subtilis 168 carrying the plasmid of PliaI + RBS + CYP2E1 + terminator/pBS0E in LB + Amp (100 μg/ml)O/N at 37°C, shaking at 170 rpm, supplemented with thiamine 1mM and 75mg/L of 5-aminolevulinic acid
↓ transfer 3 ml to 50 ml LB+Antibiotics with all the supplements in 250 ml flask
↓ measure OD650
↓ shake at 200rpm,37°C until OD650 between 0.5~0.7
↓ add 50 μl of 30ug/ml Bacitracin for induction at 25°C, shaking at 100 rpm for 18.5 hr

Reference

  1. Banerjee AL1, Swanson M, Mallik S, Srivastava DK. “Purification of recombinant human carbonic anhydrase-II by metal affinity chromatography without incorporating histidine tags.” Protein Expr Purif. 2004 Oct;37(2):450-4. doi: 10.1016/j.pep.2004.06.031
  2. Pritchard, Michael P., et al. “A General Strategy for the Expression of Recombinant Human Cytochrome P450s InEscherichia ColiUsing Bacterial Signal Peptides: Expression of CYP3A4, CYP2A6, and CYP2E1.” Archives of Biochemistry and Biophysics, vol. 345, no. 2, 1997, pp. 342–354., doi:10.1006/abbi.1997.0265.

Bacterial Lysates Preparation

While performing a 6XHis-tagged protein purification may lead to a high cost for production, we decided to choose a more economical and practical method. We performed cell disruption by 0.1-mm glass beads, which gave us the total lysates containing CA or CYP2E1 enzymes.
↓ culture Bacillus subtilis 168 carrying the plasmid of PliaI + RBS + CA + terminator/pBS0E or PliaI + RBS + CYP2E1 + terminator/pBS0E in LB + AMP (100 μg/ml) O/N at 37°C
↓ perform the protein induction procedure
↓ centrifuge at 15000 xg
↓ discard the supernatant
↓ add 3ml of PBS buffer
↓ add 1.5ml of 0.1-mm disruption beads, 10μl of DNAse I and 30μl of PMSF
↓ vortex for 1 min, then put on ice for 30 sec, repeat 8 times.
↓ centrifuge at 15000 xg for 2 min

Reference

  1. Peter E. Vandeventer, et al. “Mechanical Disruption of Lysis-Resistant Bacterial Cells by Use of a Miniature, Low-Power, Disposable Device” J Clin Microbiol. 2011 Jul; 49(7): 2533–2539. doi: 10.1128/JCM.02171-10

CO2 Absorption Analysis

↓ set up the device above
↓ pump CO2 into the glass bottle containing algae or culture media or water
↓ add CA or not
↓ set the flow rate with flow meter
↓ turn on the LED light
↓ measure final CO2 concentration with the analyzer
↓ analyze data with Vernier LabQuest® 2 software

CYP2E1 Activity Measurement

Exp1. Phenol measurement by 4-aminoantipyrine/potassium hexacyanoferrate (III) colorimetric test

↓ prepare a 96 well plate, solution I(1% of 4-aminoantipyrine in KOH solution, pH=9~10), solution II (4% of potassium hexacyanoferrate (III), K3[Fe(CN)6]), phenol (8.3g/100ml, e.g., phenol solubility in water)
↓ add 90μl of phenol solution with a serial 10X dilution
↓ add 90μl of solution I
↓ add 45μl of solution II
↓ measure at OD580

Exp2. Examination of phenol degradation by algae

↓ prepare a 96 well plate, algae, phenol(8.3g/100ml)
↓ make a phenol serial dilution as Exp1, put 90 μl into a well
↓ incubate with 90μl of Synechococcus elongatus PCC7942 (OD730=0.5) for each well
↓ culture at 37°C for one hour, shaking at 180 rpm with light
↓ transfer 90μl to a new well, and add 90μl of solution I
↓ then add 45μl of solution II
↓ measure at OD580
*(control: add ddH2O instead of algae)

Exp3. CYP2E1 enzyme activity assay

↓ prepare a 96 well plate, CYP2E1 lysates prepared with two different cofactor combination (5-aminolevulinic acid or 5-aminolevulinic acid plus FeSO4), WT lysates as a control
↓ prepare 1.79 g/L of benzene (i.e., benzene solubility in water)
↓ add 90μl of benzene solution (10X serial dilution) to each well
↓ incubate with 40μl of CYP2E1 or WT Bacillus lysates at room temperature for 30 min
↓ transfer 90μl of the mixture to a new well
↓ add 90μl of solution I
↓ then add 45μl of solution II
↓ measure at OD580

Reference

  1. M Ettinger, C Ruchhoft, R Lishka. “Sensitive 4-Aminoantipyrine Method for Phenolic Compounds” Anal. Chem.1951; 23(12) 1783-1788. doi: 10.1021/ac60060a019
  2. Mai Miura, Kensuke Ito, Maiko Hayashi, Motowo Nakajima, Tohru Tanaka, and Shun-ichiro Ogura. “The Effect of 5-Aminolevulinic Acid on Cytochrome P450-Mediated Prodrug Activation” PLoS One. 2015; 10(7): e0131793. doi: 10.1371/journal.pone.0131793