Experiments



Experiment Protocols

pUC19 Vector Preparation

Transformation of pUC19

Materials:
  • pUC19 positive control DNA (Invitrogen)
  • One Shot™ TOP10 Chemically Competent E. coli (ThermoFisher, Cat# C404003) / DH5α Competent Cells obtained from HKABE (CUHK)
  • S.O.C Medium (ThermoFisher, Cat# 15544-034)
  • LB agar (ThermoFisher, Cat# 22700-025) plates with ampicillin (Gibco™, Cat# 11593027) added
Procedures:
  1. Mix 10 μL of DNA with 1 mL of competent cells in a polypropylene tube stored at -80°C.
  2. Incubate the tube on ice for at least 10 minutes.
  3. Heat shock the tube by exposing it to 42°C for 45 seconds.
  4. Return on ice for 2 minutes.
  5. Add 1 mL of pre-warmed SOC medium to the tube.
  6. Incubate the tube at 37°C for 10 minutes for recovery.
  7. Take 100-200 μL of the solution and spread it evenly on an ampicillin LB plate.
  8. Incubate the plate at 37°C overnight.

Purification of pUC19 Plasmids

Materials:
  • PureLink™ Quick Plasmid Miniprep Kit (ThermoFisher, Cat# K210010)
Procedures:
  • Refer to manufacturer’s manual.
  • 50 μL of elution buffer were used in all preparations.

Restriction Digestion of pUC19

Materials:
  • EcoRI-HF (NEB, Cat# R3101S)
  • PstI (NEB, Cat# R140S)
  • NEBuffer™ r2.1 (NEB, Cat# R140S)
Procedures:
Component 50 µl Reaction (µL)
DNA 20
10X NEBuffer r2.1 5
EcoRI-HF 2
PstI 2
Nuclease-free Water 21
  1. Incubate at 37 °C for 1 hour.
  2. Heat inactivation at 65 °C for 20 minutes.

Gel Purification of Digested pUC19

Materials:
  • PureLink™ Quick Gel Extraction Kit (ThermoFisher, Cat# K210012)
Procedures:
  • Refer to the manufacturer’s manual.
  • 50 μL of elution buffer were used in all preparations.

Constructs Cloning

HiFi Assembly

Materials:
  • NEBuilder® HiFi DNA Assembly Master Mix (NEB, Cat# E2621S)
Procedures:
For constitutive expression constructs:
Components (µL) amilCP cjBlue eforRed tsPurple dTomato Positive
Vector 2 2 2 2 2 10
Insert 5.7 5.8 5.6 5.7 5.8
HiFi MM 10 10 10 10 10 10
H2O 2.3 2.2 2.4 2.3 2.2
Total 20 20 20 20 20 20
For biosensor constructs:
Components (µL) pPbr MerR-pCadA pCadA pYodA pMerT pZnt dTomato (+ve)
Vector 2 2 2 2 2 2 2
Insert 6 6 6 6 6 6 6
HiFi MM 10 10 10 10 10 10 10
H2O 2 2 2 2 2 2 2
Total 20 20 20 20 20 20 20
  1. Incubate at 50°C for 1 hour.

Colony PCR

Materials:
  • M13 forward and reverse primer (100uM, Tech Dragon)
  • OneTaq® 2X Master Mix with Standard Buffer (NEB, Cat# M0482S)
Primers:
Primers Sequence
M13 forward TGTAAAACGACGGCCAGT
M13 reverse CAGGAAACAGCTATGACCATG
Procedures:
  1. Prepare mastermix of PCR reaction according to the table below:
Components X1 (µL)
Forward M13 0.5
Reverse M13 0.5
Template Colonies
2X OneTaq 12.5
H2O 11.5
Total 25
  1. Run the PCR with the condition below for 30 cycles.
Stage Time (s)
Initial Denaturation (94 °C) 30
Denaturation (94 °C) 20
Annealing (55 °C) 40
Extension (68 °C) 60
Final Extension (68 °C) 300

Gel Electrophoresis

Materials:
  • The MiniOne Electrophoresis System (MiniOne Systems, Cat #M1000)
  • Gel Loading Dye Purple (6X) (NEB, Cat #B7024S)
  • UltraPure™ Agarose (Invitrogen, Cat #16500500)
  • AE Buffer (Tris-acetate-EDTA) (50X) (ThermoFisher, Cat# B49)
  • Quick-Load® Purple 1kb Plus DNA ladder (NEB, #N0550S)
  • GelGreen™ DNA Stain 50 µl (MiniOne Systems, Cat# M3113)
Procedures:
  1. Add 5 μl of loading dye into each tube of DNA.
  2. Add 25 μl of distilled water into a new tube and 5 μl of LD into the tube (negative control).
  3. Set up the gel tank:
  4. Prepare 1X TAE buffer for gel forming and gel tank buffer.
  5. Prepare 1% agarose gel with 1X TAE buffer in a conical flask.
  6. Microwave the gel for 1 min to make the agarose dissolve completely.
  7. Cool down the conical flask with running water.
  8. Add Gel Stain into agarose gel for DNA visualization (i.e. 2 μL in 20mL of gel).
  9. After the gel forms, transfer it into the gel tank and add buffer to just cover the whole gel.
  10. Add the DNA and ladder into the wells.

E. coli Culturing

E. coli Growth Media

Materials:
  • LB Broth Base (Lennox) (Invitrogen, Cat# 12780052)
  • LB Agar (Lennox L Agar), powder (Invitrogen, Cat# 22700025)
  • Ampicillin, sodium salt, irradiated (Gibco™, Cat# 11593027)
Procedures:
  1. Prepare the media following the manufacturer's instructions using distilled water.
  2. Sterilize the media using a pressure cooker (since we don't have an autoclave).
  3. After cooling down, add ampicillin.
  4. Unless otherwise noted, incubate the cultures at 37°C, 180 rpm.

Functional Assay of Heavy Metals

Heavy Metal Functional Assays

Materials:
  • Lead (II) Nitrate solution (2 mM)
  • Cadmium (II) Chloride solution (4 mM)
  • Mercury (II) chloride solution (2 mM)
  • Zinc (II) chloride solution (4 mM)
Procedures:
  1. Add 1 mL of the metal solution to 19 mL of LB/Ampicillin broth with the cultures.
  2. Incubate for at least 12 hours at 37°C, 180 rpm.
  3. Centrifuge 1 mL of the culture for 2 minutes at 8,000 g.
  4. Observe the colour change in the pellet.

Lead Concentration-Dependent Response Assay

Materials:
  • Lead (II) Nitrate solution (2 mM)
Procedures:
  1. Dilute the stock metal solution to the desired concentration.
  2. Add 1 mL of the metal solution to 19 mL of LB/Ampicillin broth with the cultures.
  3. Incubate for at least 12 hours at 37°C, 180 rpm.
  4. Centrifuge 1 mL of the culture for 2 minutes at 8,000 g.
  5. Observe the colour change in the pellet.