Experiment protocols

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Preparation of pUC19 vector for Hifi Assembly of gBlock fragments

Transformation of pUC19

Materials:

pUC19 positive control DNA (Invitrogen), One Shot™ TOP10 Chemically Competent E. coli (ThermoFisher, Cat# C404003), S.O.C Medium (ThermoFisher, Cat# 15544-034), LB agar (ThermoFisher, Cat# 22700-025) plates with ampicillin added

Procedures:

1. Mix 5 μL of DNA with 1 mL of competent cells in a polypropylene tube stored at -80°C.
2. Incubate the tube on ice for 5 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 30 minutes for recovery.
7. Take 100 μL of the solution and spread it evenly on an ampicillin LB plate.
8. Incubate the plate at 37°C overnight.

Preparation of pUC19 plasmids

Materials:

PureLink™ Quick Plasmid Miniprep Kit (ThermoFisher, Cat# K210010)

Procedures:

Refer to manufacturer’s manual.

Restriction digestion of pUC19

Materials:

EcoRI-HF (NEB, Cat# R3101S), CutSmart Buffer (NEB, Cat# B6004S)

Procedures:

Gel purification of digested pUC19

Materials:

PureLink™ Quick Gel Extraction Kit (ThermoFisher, Cat# K210012)

Procedures:

Refer to the manufacturer’s manual.

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Cloning recombinant plasmids and introduce into host cells.

Hifi assembly

Materials:

NEBuilder® HiFi DNA Assembly Master Mix (NEB, Cat# E2621S)

Procedures:

Refer to manufacturer’s manual.

Heat shock transformation of recombinant plasmid

Materials

One Shot™ TOP10 Chemically Competent E. coli (ThermoFisher, Cat# C404003), S.O.C Medium (ThermoFisher, Cat#15544-034)

Procedures:

1. Combine 5 μl of recombinant plasmid with 1 ml of competent cells in a polypropylene tube stored at -80°C.
2. Place the polypropylene tube on ice and incubate for 5 minutes.
3. Conduct a heat shock by exposing the tube to 42°C for 45 seconds.
4. Add 1 ml of SOC medium to the polypropylene tubes.
5. .Incubate the tubes at 37°C for 30 minutes to facilitate recovery.
6. Add 100 μl of the solution to an ampicillin plate.
7. Spread the solution evenly across the plate and incubate at 37°C to promote bacterial growth.

Verification of the assembly

Double enzyme digestion

Materials:

EcoR1-HF enzyme(NEB, #R3101S) , Xbal enzyme(NEB, #R0145S), CutSmart Buffer (NEB, Cat# B6004S)

Procedures:

1. Prepare reaction mixture according to the table below:
   Components

   1X (µl)

   EcoRI-HF

   0.5

   XbaI

   0.5

   DNA (~0.5 ug/µl)

   2

   10X CutSmart Buffer

   2.5

   dH2O

   19.5

   Total Volume

   25
2. Incubate at 37oC for at least 1 hour.

Colony polymerase chain reaction (PCR)

Materials:

M13 forward and reverse primer (100uM, Tech Dragon), OneTaq® 2X Master Mix with Standard Buffer (NEB, Cat# M0482S)

Procedures:

1. Prepare mastermix of PCR reaction according to the table below:
   Components

   1X (µl)

   Forward Primer (10 µM)

   0.5

   Reverse Primer (10 µM)

   0.5

   Template DNA

   5

   OneTaq 2X MasterMix

   12.5

   dH2O

   6.5

   Total Volume

   25
2. Run the PCR with the condition below for 25-30 cycles:
   Stage

   Time (s)

   Initial Denaturation (94°C)

   300

   Denaturation (94°C)

   25

   Annealing (55°C)

   55

   Extension (68°C)

   90

   Final extension (68°C)

   300




Gel electrophoresis

Materials:

Gel Loading Dye Purple (6X) (NEB, #B7024S), Agarose (invitrogen ,#LOT0000695970) , 50 X TAE buffer (ThermoFisher, #LOT 00671729), Quick-Load® Purple 1kb Plus DNA ladder (NEB,#LOT10034620)

Procedures:

1. Add 5μl of loading dye (LD) into each tube of DNA
2. Add 25μl of distilled water into a new tube and 5μl 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 / TBE 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 uL in 20mL of gel)
9. After the gel formed, transfer the gel into the gel tank and add buffer to just cover the whole gel
10. Add the DNA and ladder into the wells.



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Functional assay of formaldehyde

Materials:

40% Formaldehyde solution (methanal) LB broth (ThermoFisher)

Procedures:

1. Dilute 40% formaldehyde to desired concentrations with LB broth solution
2. Inoculate 10 mL of LB/Ampicillin broth with E. coli with formaldehyde solution to concentrations specified (500 uM and 1000 uM)
3. Incubate for 12 hours
4. Centrifuge the culture for 3 minutes at 6,000 g
5. Observe any color change in the pellet