iGEM Uppsala 2022 - General Protocols

These are the protocols that have been used throughout this project, unless otherwise stated. If another page links to this one, that means that one of the following protocols were used. If any work was done that diverged from these protocols, it is described in the section of that work in detail.

Agarose gels can be prepared at different agarose percentages, as a 2% agarose gel allows for the separation of smaller fragments than a 1% agarose gel, as such this protocol should be adapted according to the sample.

Mix agarose with 1XTAE buffer to a concentration of 1 or 2% (e.g: to prepare 200mL of 1% agarose gel, add 2mg of agarose to 200mL of 1XTAE buffer).
Heat the solution in a microwave and watch as the solution heats up.
When bubbles start to form, pause the heating process, mix solution for a few seconds and proceed, stopping each time bubbles start to form.
When the solute is no longer visible, the gel can be cast, by pouring it into its mold.
Let it cool down and solidify at room temperature.

There are many ways to prepare competent cells. One such way is through the use of CaCl₂, which keeps the cell wall more permeable, facilitating the entrance of circular DNA.

Make an overnight culture of E. coli cells, keeping it at 37ºC, with shaking.
Dilute overnight culture 1:100 in SOB medium.
Measure OD600 and let the culture grow at 37ºC with shaking until and OD600 of 0.4 is reached.
Incubate cells on ice for 15min.
Centrifuge at 3500rpm, at 4ºC for 5min.
Discard the supernatant.
Resuspend pellet with a micropipette, in 100microliters of 0.1M CaCl₂, kept at 4ºC before use.
Incubate the cells on ice for 30min.
Centrifuge at 3500rpm, at 4ºC for 5min.
Resuspend the cells in 2 mL ice-cold 0.1 M CaCl₂/20% glycerol.
Incubate the cells for 45min on ice.

After this, you may wish to keep your cells in a glycerol stock, so as to be able to use them later. To do so, follow the next steps:

Snap freeze the cells with liquid nitrogen.
Store at -80ºC.

Day 1:

Inoculate single colony into 2mL LB/antibiotic media, grow 37℃, 220rpm overnight.

Day 2:

Inoculate the O.N culture 1:100 into LB/antibiotic media.
Shake at 37℃, until desired OD600, add desired concentration of inducer directly into the culture (same volume of dH₂O in uninduced controls), put in desired temperature, 220rpm shaking, start induction for desired time.
After induction, harvest and lyse the culture by either sonication or SDS (not for purification).

For Purification also do:

After sonication, centrifugate cell lysates at 14000 rpm for 10 min at 4 °C, collect supernatants and filter through 0.2 μm filters and keep on ice.
Nickel ion column purification
1. Equilibrium the column by 3x volume Buffer A
2. Load the sample onto the column (can repeat load the flow through again if you want)
3. Wash the column with Buffer A 20mL (or 5x volume)
4. Elute with Buffer A containing 20mM, 50mM, 100mM, 250mM, 500mM imidazole. 16mL each concentration. Collect the flow through by Eppendorf tube, 1mL/tube (normally the protein will be eluted at 250mM and 500mM, but collect all concentrations at first time), put on ice.
5. Wash the column with 5x volume Buffer A containing 500mM imidazole.
6. Wash the column with 5x volume of ddH₂O.
7. Wash the column with 5x volume Buffer A, half fill the column, keep at 4℃.
Run SDS-PAGE to check the purification results.
Concentrate the protein if needed.
Mix protein with 50% glycerol (final conc.), freeze at -80℃.

Buffers:

5x stock buffer (no need for adjusting pH & filtration): 250mM Tris-HCl, 2.5M NaCl.

Sonication Buffer: 50 mM Tris-HCl pH 8.0 containing 500 mM NaCl (same as buffer A, check your protein’s pH).

Buffer A: 50mM Tris-HCl, 500mM NaCl, pH=8.0 (check your protein’s pH).

Buffer B: 500mM imidazole, 50mM Tris-HCl, 500mM NaCl, pH=8.0 (check your protein’s pH).

Mix Buffer A and B to get buffer A containing different imidazole concentrations, filtrate the buffers through 0.2 μM filter before using. Cool down the buffer at 4℃ if needed.

(Protocol modified from GeneArt™ Gibson Assembly® HiFi Master Mix from Thermo Fisher Scientific)

Assembly mix: Mix 0.08 pmol of each DNA insert and the vector. Double acquired volume with the provided 2X GeneArt™ Gibson Assembly® HF Master Mix. Add dH₂O up to 20 μL.
Positive control: Mix 10 μL of the provided positive control with 10 μL 2X GeneArt™ Gibson Assembly® HF Master Mix.
Gently vortex the samples and spin down at 4000 rpm for 15s.
Incubate at 50 ^oC for 15 min
Screening: Transform cells and plates with proper antibiotics (positive control on kanamycin and ampicillin).

Mix 20 ng of each digestion mixture.
Add 2 μl of 10x reaction for T4 DNA ligase.
Add 1 μl of T4 DNA ligase.
Add ddH₂O to a final volume of 20 μl.
Incubate at room temperature (~22°C) for 30 min.
Heat-inactivate the enzymes at 80°C for 20 min.

Samples may be stored at -20°C.

Prepare O/N cultures in LB media with the appropriate antibiotic, at 37ºC with shaking, ideally at the end of the day so as to keep them as fresh as possible.
The following day inoculate the O/N culture into fresh LB media with antibiotic, to a 1:100 concentration.
Measure OD600 at the beginning and again approximately every 20min until OD600 reaches 0.6-0.8, depending on when the cells should be induced. Measurements become less accurate when absorbance passes 0.5, so samples should be diluted before each measurement, by diluting them 1:10 in the same media. When an OD600 of 0.6-0.8 is reached, the culture should be induced.

Touchdown PCR:

Step		Temperature (°)	Time
Initial denaturation		98	1 min
30 cycles	Denaturation	98	20 s
	Annealing	72 (2 cycles)	45 s
		71 (2 cycles)
		70 (2 cycles)
		69 (2 cycles)
		68 (22 cycles)
	Extension	72	40 s or 75 s
Final extension		72	7 min
Hold		4	---

PCR programme for gradient and limited gradient:

Step		Temperature (°)	Time
Initial denaturation		98	1 min
30 cycles in gradient (10 cycles in limited gradient)	Denaturation	98	20 s
	Annealing	72 (back) - 64 (front)	45 s
	Extension	72	40 s or 75 s
Final extension		72	7 min
Hold		4	---

All PCR reactions were performed in 50 μl, containing 25 μl of Q5 high fidelity 2x Master Mix from NEB, 0.25 μl of forward primer, 0.25 μl of reverse primer, 1 μl of template DNA (1 ng/μl) and 23.5 μl of ddH₂O.

PCR reaction mixture for touchdown, gradient and limited gradient:

Component	Volume (1 rxn = 50 μL)
ddH₂O	23.5 μl
Q5^® High-Fidelity 2X Master Mix	25 μl
Forward primer 10 μM	0.25 μl
Reverse primer 10 μM	0.25 μl
Template DNA (1 ng/μl)	1 μl

Colony PCR programme:

Each tube for PCR reaction contained 4 μl of 5x Phusion HF buffer (Thermo Scientific), 0.4 μl of 10 mM dNTPs, 0.5 μl of forward primer, 0.5 μl of reverse primer, 14.4 μl of ddH₂O, 0.2 μl of Phusion High-Fidelity DNA Polymerase (Thermo Scientific) and a scraped piece of desired colony as template. Total volume was 20 μl.

Step		Temperature (°)	Time
Initial denaturation		98	30 s
30 cycles in gradient (10 cycles in limited gradient)	Denaturation	98	7 s
	Annealing	68	30 s
	Extension	72	Based on desired product length
Final extension		72	7 min
Hold		4	---

PCR reaction mixture for colony PCR.

Component	Volume (1 rxn = 20 μl)
ddH₂O	14.4 μl
5X Phusion^TM Buffer (Thermo Scientific)	4 μl
10 mM dNTPs	0.4 μl
Forward primer 10 μM	0.5 μl
Reverse primer 10 μM	0.5 μl
Template DNA	Scraped piece of desired colony
Phusion^TM High-Fidelity DNA polymerase (Thermo Scientific)	0.2 μl

PCR Purification was done using; “Protocol A. DNA purification using centrifuge”, provided in ThermoFisher Scientific's GeneJET PCR Purification Kit.
Download manual PDF or View on ThermoFisher's website

For the enzymatic assay of PETase activity two different types of pretreatment of PET were used, DMSO and triton treatment.

Samples with DMSO contained 12 ml of cell lysate and 3 ml of DMSO Mix (42 % (v/v) DMSO, 250 mM glycine-NaOH (pH 9), 250 mM NaCl).

Triton treatment was derived from the TJUSLS China 2021 iGEM team. PET used in samples treated with triton was incubated for 30 minutes in 50 °C in a mixture containing 0.5% (v/v) Triton X-100 and 10 mM Na2CO3. PET was then air-dried in 37 °C and put in samples containing 12 ml of cell lysates and 3 ml of glycine NaOH (pH 9).

Samples were incubated at different conditions and with different types of PET (see results) and samples with uninduced PETase cells and with induced cells expressing IF3 protein were incubated in all the stated conditions and used as negative controls.Samples for measurement were taken every few hours in the course of 3 days and every time sample was taken, heat inactivated at 85 °C for 10 mins and centrifuged (4000 rpm, 5 mins) to dispose of cell debris and PET powder and measured spectrophotometrically.

Make 3 mL O/N cultures (with appropriate antibiotic for the current bacteria). Amounts found below:
1. 12 µg/mL tetracycline
2. 100 µg/mL ampicillin
3. 25 µg/mL chloramphenicol
4. 50 µg/mL kanamycin
Extract plasmids with “GeneJET PCR Purification Kit” from Thermo Scientific (see protocol 11 for link to download)

To two separate tubes, add 500 ng of DNA of each sample (one sample being plasmid containing target sequence, the other sample being the backbone said target sequence will later be ligated into).
Add 5 μl of 10x reaction buffer compatible with both enzymes, to both DNA mixtures separately.
Add 1 μl of each of the endonucleases to each mixture.
Add ddH₂O to a final volume of 50 μl.
Tap on the tubes to mix. Centrifuge for a few seconds to spin down liquid.
Incubate at 37°C for 30 min.
Heat-inactivate the enzymes at 80°C for 20 min.

Samples may be stored at -20°C.

Running buffer (10X):

Component	Amount
Tris	30.3 g
Glycine	144 g
SDS	10.0 g
dH₂O	1000 mL

Loading/sample buffer (5X):

Component	Amount
Tris-HCl pH 6.8	250 mM
SDS	10 % (w/v)
Glycerol	25 % (v/v)
DTT	100 mM
Bromophenol blue	0.5 % (w/v)

Gel recipie for X% acrylamide gel:

Component	Amount for resolving gel	Amount for stacking gel
Acrylamide, 30%	(0.5 x X) mL	1.98 mL
Tris, 0.5 M pH 6.8	-	3.78 mL
Tris, 1.5 M pH 8.8	3.75 mL	-
SDS, 10% w/v	150 μL	150 μL
H₂O	11.02 - (0.5 x X) mL	9 mL
TEMED	7.5 μL	15 μL
APS, 10% w/v	75 μL	15 μL
---Total volume---	15 mL	15 mL

Harvest and lysis by sonication:

Harvest cells by centrifugation at 4℃, 4000rpm. Discard the supernatant, resuspend the cell pellet in sonication buffer (10mL cell culture in 2mL buffer, for 1L culture, use 20mL buffer).
Lyse by sonication (cycles of sonication for 5 s and pausing 5 s, with total time of 40 min) on ice-water mixture.

Harvest and lysis by SDS:

Transfer 200 μL of the liquid cultures to a microcentrifuge tube and spin down at 6000 rpm for 4.5 min.
Resuspended the pellets in 50 μL 1X SDS sample buffer.
Incubated samples at 95 oC or 10 min and then spin down at 10 000 rpm for 10 min.

Fill a chamber with 1x running buffer.
Put gel into a casette (symmetrically) and then close the chamber.
Run at 200 V for ~40 min or until the stain has almost reached the bottom of the gel.

Standard version

Transfer the gel to a suitable container and add staining solution (should cover gel). Let sit overnight.
Start the destaining by pouring out the staining solution and adding destaining solution. Pour that out and then add new destain.
Put gel with destaining solution on shaker and replace the solution as it gets dyed.

The gel can then be stored in the destain overnight.

Quick version

Staining solution is added to the gel and microwaved for 30 s and then incubated on a shaker for 20 min,
Wash the gel with dH₂O and then add ~30 mL destaining solution. Microwave for 45 s and then incubated on a shaker for ~20 min. This step is repeated until a desired destain was achieved.
Do a final wash of the gel with dH₂O.

Recommended: Digesting 0.2-0.15 μg of DNA with a 2-fold to 10-fold excess of enzyme in a total volume of 20 μl. A typical restriction enzyme digestion protocol:

Add following reaction components in the order indicated:

Water, nuclease-free	16-16.5 μl
10X recommended buffer for restriction enzyme	2 μl
Substrate DNA	1 μl (~1 μg)
Restriction enzyme	0.5-1 μ (5-10 u)
---Total volume---	---20 μl---