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Experiments

Describe the experiments, research and protocols you used in your iGEM project.

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• Protocols
• Experiments
• Documentation of the development of your project

Protocols

Heat shock competent cells

Preparation

Day 1 Inoculate cells in 3.5mL LB medium. Incubate at 37°C overnight.

Day 2 Measure culture OD at 600nm. Dilute cells in 250mL of LB medium so OD equals to 0.12. Incubate overnight at 20°C and 180rpm.

Day 3 Measure culture OD at 600nm and dilute to obtain OD_600nm=0.6. Cells must be kept at 4°C during all following steps. Put on ice for 10min and centrifuge for 10min at 3000rpm and 4°C. Discard supernatant and resuspend cells in 80mL of fresh TB buffer. Keep on ice for 10min and centrifuge for 10min at 3000rpm and 4°C. Discard supernatant again and resuspend cells in 20mL of fresh TB buffer with 7% of DMSO. Keep on ice for 10min. Aliquot cells and freeze with liquid nitrogen. Keep at -80°C.

TB buffer recipe

HEPES	10mM
MnCl2	55mM
CaCl2	15mM
KCl	250mM
KOH

Dissolve HEPES, CaCl₂ and KCl in water. Adjust pH to 6.7 with KOH. Add MnCl₂. Filter to sterilize and keep at 4°C.

Tranformation

Add 1µL of plasmids to 50µL of competent cells (make a control tube without plasmid). Keep tubes on ice for 30min and heat shock at 42°C for 1min. Add 500µL of LB medium into each tube and incubate at 37°C for 1h. Spread cells on Petri dishes in duplicate and incubate at 37°C overnight.

Electro-competent cells

Preparation

Inoculate 15mL of LB with 200µL of an overnight cell culture. Incubate at 37°C and 180rpm until OD_600nm reaches 0.6. Centrifuge cells for 10 minutes at 4000rpm. Wash twice with 10mL of 10% glycerol. Put cells in 200µL of 10% glycerol and use for electroporation.

Transformation

Add 1µL of plasmids to 50µL of competent cells (make a control tube without plasmid). Electroporate (the variable TODO??? should be close to 6) and recover cells with 1mL of cold LB medium. Incubate cells for 1h at 37°C. Spread cells on Petri dishes in duplicate and incubate at 37°C overnight.

Plasmid DNA extraction

Transfer 1.5mL of an overnight culture into an eppendorf tube. Centrifuge at 13000rpm for 1min to pellet the cells. Discard supernatant and resuspend cells in 100µL of TE buffer. Add 200µL of solution II and mix gently by inverting the tubes until lysate appears clear. Add 150µL of solution III and mixed gently. Keep the solution on ice for 10min. Centrifuge for 10min at 13000rpm and recover the supernatant. Add 100µL of phenol in each tubes to denature the proteins and vortex for 30s. Centrifuge the tubes for 7min at 13000rpm. Recover the aqueous phase. Add 2 volumes (900µL) of 100% ethanol and put at -20°C for 10min. Centrifuge the tubes again for 10min at 13000rpm. Discard supernatant and wash with 800µL of 70% ethanol to remove the remaining ions. Make sure that the pellet containing DNA remains at the bottom of the tubes. Centrifuge the tubes for 4min at 13000rpm and remove supernatants. Dry tubes in speedvac. Resuspend the pellet in 50µL of TE/RNAse. Keep the extracted plasmids at -20°C.

TE buffer recipe

Tris	10mM
EDTA	55mM

Solution II recipe (lysis solution) Keep for maximum 3 weeks.

SDS	1%
NaOH	0.1M

Solution III recipe (neutralization solution) CH₃COOK 3M at pH4.8

CH3COOK 5M	60mL
CH3COOH	11.5mL
water	28.5mL

TE/RNase Use only fresh solution.

RNase (10mg/mL)	5µL
TE	1mL

DNA extraction using the Invitrogen ChargeSwitch®-Pro Plasmid Miniprep Kit

Follow the steps below to purify plasmid DNA from 1-5ml of fresh overnight cultures using a microcentrifuge. All steps are performed at room temperature.

Before Starting

• For a new kit, add Rnase A (provided in the kit) to the Resuspension Buffer and mix.
• If necessary, warm the Lysis Buffer to 37°C to dissolve any precipitate.

Preparing the Sample

1. Pellet cells from 1-5 ml of overnight culture.
2. Resuspend in 250 µL of Resuspension Buffer premixed with Rnase A. No cell clumps should remain.
3. Add 250 µL of Lysis Buffer. Mix well by gentle inversion. Do not vortex.
4. Incubate at room temperature for 2-5 minutes. Do not incubate more than 5 minutes.
5. Add 250µL of Precipation Buffer, and mix well until a white precipitate is formed.
6. Centrifuge for 10 minutes at maximum speed to pellet the debris.

Binding the DNA

1. Carefully transfer the supernatant from Step 2.6 above onto the ChargeSwitch®-Pro MiniPrep Colum inserted in a Collection Tube.
2. Centrifuge the column/tube at maximun speed for 30-60 seconds.
3. Remove the column from the tube and discard the flow-through. Re-insert the column in the same tube.

Washing the Column

1. Add 750µL of Wash Buffer 1 to the column.
2. Centrifuge the column/tube at the maximun speed for 30-60 seconds.
3. Remove the column from the tube and discart the flow-through. Re-insert the column in the tube;
4. Add 250µL of Wash Buffer 2 to the column.
5. Centrifuge the column/tube at maximun speed for 30-60 seconds.
6. remove the column from the tube. Discard the flow-through and the Collection Tube.

Eluting the DNA

1. Insert the column into an Elution Tube (provided in the kit).
2. Add 50-100µL of Elution Buffer onto the column.
3. Centrifuge the column/tube at maximun speed for 30-60 seconds.
4. Optional:Remove the Elution Tube and transfer the eluate back onto the same column. Re-insert the column in the tube and centrifugate at maximun speed for 30-60 seconds. This step is recommended for maximun recovery.
5. The eluate contains your purified plasmid DNA. Store DNA at 4°C or -20°C.

Plasmid digestion

Mix 10µL of extracted plasmid with 2µL of digestion buffer and 7µL of water. Add 1µL of restriction enzyme. Incubate for 1 hour at 37°C. Mix 5µL of DNA with 5µL of water and 2µL of DNA loading buffer. Migrate on an agarose gel.

gBlock plasmid insertion

The following ratio should be over 7: \[ \frac{vector\ size}{insert\ size}\times\frac{insert\ concentration}{vector\ concentration} \]

Gently centrifuge gBlocks. Add 100µL of TE buffer (final concentration should be around 10ng/µL). Vortex and incubate at 50°C for 20min. Mix 7µL of gBlocks with 1µL of plasmid extracted as described earlier and 1µL of buffer. Incubate for 1h at RT.

DNA electrophoresis on agarose gel

Gel preparation

Mix agarose and TAE buffer to get a final concentration of 0.8%(w/v). Microwave for 1-2min in order to melt the agarose. Let cool down for 10min and add BET up to 5µg/mL. This solution can be kept at 61°C. Pour the solution into the casting tray. Place the comb and let cool down for about 30min. Place the gel in an electrophoresis unit full of TAE and load the samples plus a ladder. Migrate at 100V for 20min. Visualize DNA fragments with UV light.

Polymerase chain reaction

PCR with Taq PCRx DNA Polymerase (recombinant) or DreamTaq DNA Polymerase from ThermoFisher Scientific

• Prepare enough PCR master mix for the number of colonies analyzed plus one extra. For each 20μl of reaction, mix the following reagents :

Components	Volume
10x Taq buffer with KCl	2µL
MgCl2 (25mM)	1.2µL
dNTP (10mM)	2µL
Primers mix (10µM each)	1.2µL
Taq DNA polymerase	0.1µL
Nuclease-free water	up to 20µL
Total volume	20µL

• Mix gently and aliquot 20μl of the mix into the PCR tubes on ice.
• Pick up an individual colony with a sterile pipette tip and resuspend it in 20μL of the PCR master mix (or take 1μL of liquid preculture). Make a short strike with the same tip over culture plate to save the clone for repropagation.
• Perform PCR as follow (Tm can be calculated here):

Step	Temperature	Time
Initial denaturation	95°C	3min
30 cycles	94°C	30sec
	Tm	30sec
	72°C	1min/kb
Final Extension	72°C	7min
Hold	4°C	$\infty$

• Analyze on a gel for the presence of the PCR product of the expected length.

PCR with Q5^® High-Fidelity 2X Master Mix from NEB

• Prepare enough PCR master mix for the number of colonies analyzed plus one extra. For each 25μl of reaction, mix the following reagents :

Components	Volume
Q5 High-Fidelity 2X Master Mix	12.5µL
Primers mix (10µM each)	2.5µL
Template DNA (from extractions)	1µL
Nuclease-free water	up to 25µL
Total volume	25µL

• Mix gently and aliquot 25μl of the mix into the PCR tubes on ice.
• Perform PCR as follow (Tm can be calculated [http://tmcalculator.neb.com/#!/ here]):

Step	Temperature	Time
Initial denaturation	98°C	3min
30 cycles	98°C	10sec
	Tm	20sec
	72°C	30sec/kb
Final Extension	72°C	2min
Hold	4°C	$\infty$

• Analyze on a gel for the presence of the PCR product of the expected length.