Methods

Gibson

Protocol used for Gibson

Modifications

_insert

_vector

_gibson

_water

Transformation

E.coli TOP10

Chemical Transformation Procedure

Modifications

E.coli DH5Alpha

Protocol

PCR

Tag polimerase (25µl reaction) Used for colony PCR for cyc1

Protocol Here

Specifications

10X Standard Taq Reaction Buffer	2.5 μl
10 mM dNTPs	0.5 µl
10 µM VF2	0.5 µl
10 µM VR	0.5 µl
Template DNA	variable
Taq DNA Polymerase	0.125 µl
Nuclease-free water to	25 µl

1) 95°C	30 sec
2) 95°C	30 sec
3) 63°C	1 min
4) 68°C	1 min/kb
5) 2/30X
6) 68°C	5 min
7) 10°C	forever

Phusion polymerase (20µl reaction) Used for colony PCR for nmt1 and composite part :

Protocol Here

Specifications

15X Phusion HF or GC Buffer	4 µl
10 mM dNTPs	0.4 µl
10 µM VF2	1 µl
10 µM VR	1 µl
Template DNA	variable
Phusion DNA Polymerase	0.2 µl
Nuclease-free water to	20 µl

1) 95°C	30 sec
2) 98°C	30 sec
3) 63°C	1 min
4) 72°C	1 min/kb
5) 2/30X
6) 72°C	5 min
7) 10°C	forever

5 PRIME HotMasterMix (50µl and 10µl reaction) Used for colony PCR for nmt1 and composite part :

Protocol Here

Specifications

10 µM VF2	0.4 µl
10 µM VR	0.4 µl
Template DNA	variable
Phusion DNA Polymerase	4 µl
Nuclease-free water to	10 µl

1) 92°C	2 min
2) 94°C	20 sec
3) 55°C	30 sec
4) 70°C	1 min/kb
5) 2/30X
6) 70°C	5 min
7) 10°C	forever

Gel

Weigh out 0.5 g of agarose and mix it with 50 ml of 1x TAE buffer in a 100 ml Erlenmeyer flask.

Dissolve the agarose by bringing the mixture to the boiling point in a microwave oven, followed by mixing (by swirling the flask). Repeat the heating and mixing until all the agarose has dissolved.

Cool the agarose solution to ~50 o C by leaving it on the bench for ~20 min (or you may accelerate the cooling by applying cold water from the tap to the outside of the flask).

Using gloves, add 5 l GelRed (10 000x). Swirl the flask gently to mix, try to avoid bubbles.

Pour the gel carefully into the mold. Bubbles may be removed/punctured by using a pipette tip.

Miniprep

Protocol

Modifications:

During the first attempt ethanol was not added to the PE buffer, which resulted in an unsuccessful miniprepl

In the second attempt 72/4% ethanol was added, as opposed to the recommended 96-100%, resulting in a successful miniprep

French press

Protocol

Load cells into Cell:

Attach flow valve to big straight hole. Look at the nylon ball at he end and make sure it’s not distorted and misshapen. Make sure valve is closed. Attach dispenser + small tubing to the other hole (small and slanted).
Examine the piston to make sure the o-rings are not nicked or distorted. Push piston into cell to line.
Remove cap on other end.
Pour sample into cell. Fill completely (1 mL for small cell).
Put cap on the end. Firmly push the cap down – the best way is to use the heel of your hand and hit the cap hard until it’s completely seated.
Turn setup over. Put in Press with piston up.
Mini cell can be loaded in your hand, but the large cell is too heavy to load this way. There is a black 3 columned stand next to the Press (may be pushed back on the counter). Put the large cell upside down (piston down). Fill the cell the same way as the mini cell.

Run:

Turn the Ratio Selector to Down position and turn the Pressure Increase control fully counterclockwise. The press needs to go down enough for the cell and extended piston.
Pump on.
Pressure Increase clockwise to 800 (turn knob) for mini-cell and 1000 for large cell.
Pump off.
For mini-cell turn Ratio Selector to medium.
Put cell on the stand – make sure you can turn the flow valve on the cell, that it’s not blocked by anything. Make sure the cell is aligned properly so that the piston squarely strikes under the upper platen. Make sure the piston handles are perpendicular to the bar and it’s screws (if it’s not, as the piston is pushed down the handles will run into the screws and something will break). Swing the bar across the cell and make sure it is completely against the cell. If it’s not, the cell could pop off the stand when pressure is applied. If the screws on the bar get in the way – unscrew them enough to slide over cell and then tighten them).
Pump on.
When the pressure gets to 800, slowly release cells by tapping (not hitting) the flow valve (black handle) with a pen – cells must come out slowly, drop by drop (pump on still), 15 drops/min. Several labs feel that tapping with a pen gives a more consistent and reproducible release of the cells. The drop rate tends to increase near the end of the run. As you approach the end of the run, you may want to close the flow valve slightly by turning it clockwise before opening it again. Also, there might be air bubbles in the sample and these tend to squirt into the collection tube and if you aren’t careful where you hold your collection tube, you could lose your sample. Be very careful that the tubing is in your collection tube and not pointing towards your face.
Pump off.
Turn the Ratio Selector down.
If you are done for the day, turn the Pressure Increase control fully counterclockwise.
Pump on.
Wash cell with H₂O.

sfGFP purification

Agnieszka Wrobel

Materials

pASK-IBA3 sfGFP (Amp100)
BL21 (DE3) Gold competent cells (50µl per sample)
1.5mL Microtubes
SOC Media (950µL per sample)
Petri plates w/ LB agar and ampicillin (100µg/ml) (2 per sample)

Following buffers were made (and sterilized):

Running buffer (per 1 L) (40 mM sodium phosphate (7,11g), 400 mM NaCl (23,4g), 20 mM imidazole (1,36g), pH 8.0 – made 2L
Elution buffer (per 1 l)(40 mM sodium phosphate (7,11g), 400 mM NaCl (23,4g), 500 mM imidazole (34g), pH 8.0 – made 1L
1 M MgCl2 – used 20µl
1M MnCl2 - used 20 µL
LB medium – 2L

Equipment

Floating Foam Tube Rack
Ice & ice bucket
Lab Timer
42°C water bath
37°C incubator
Sterile spreader or glass beads
Pipettes and tips (10µL, 20µL, 200µL, 1000µL)
Microcentrifuge
Pipetboy controller
ÄKTA start (chromatography)
Frac30 fraction collector
French Pressure Cell

Method

Day 1

Single Tube Transformation Protocol

The cells that we used were E. coli BL21 Gold
The plasmid that we used to transform the cells was pASK-IBA3 sfGFP (Amp100) (glycerol stock 709)
This plasmid was not resuspended as it was already dissolved in water.
The incubation after transformation was done at 220rpm.
We did not make the overnight culture and did not run the PCR (points 15. and 16. In the protocol).
We let the cells grow on plates at 37ºC until the next day (19 plates in total).

Day 2

We made 10 ml of an overnight culture of E.coli BL21 Gold (pASK-IBA3 sfGFP) Amp100 from each petri plate.
LB medium for the next day was already prepared by the researches at the laboratory (2L).

Day 3

We inoculated 800 mL of LB/Amp100 (100µg/ml) with 5 mL of the overnight culture of E.coli BL21 (DE3) Gold (pASK-IBA3 sfGFP) Amp100 (x 2 bottles)
We grew the cells until OD600=0.5 at 37ºC. This took us 3 hrs.
We prepared 12% SDS-PAGE gel.
Induce the cells with AHTC 0.2 µg/mL
Then we grew cells for another 2 hours at 30ºC
We then spun the cells: 4500 rpm, 20 min, RT
The pellet was resuspended in 20 mL running buffer
The cells were frozen at -80ºC.

Day 4

Cells in the tubes were thawed.
Protease inhibitor was added (1 tablet, thermos scientific – prod. 88266)
1 mM MgCl2 and 1 mM MnCl2 was added as well (20 µL of 1M stock/ per 20 mL)
20 µL of DNaseI solution (10mg/mL) was added to each tube (10 µg/mL)
200 µL of Lysozyme solution (10mg/mL) was added to each tube (0.1 mg/mL)
French press: The samples were kept on ice at all times. We took out 30 µL of the protein sample to run it on a 12% SDS-Page gel. ( Protocol for French Press )
Ultracentrifuged 20 000 g, 35 min, 4 C. Balance tubes.
For affinity chromatography we used supernatant. We took out 30 µL of the protein sample to run it on a 12% SDS-Page gel.
Filter sterilize the supernatant through the 0.2 uM filter before applying to the column.
Affinity chromatography:

Running buffer:

40 mM sodium phosphate
400 mM NaCl
20 mM imidazole
pH 8.0

Elution buffer:

40 mM sodium phosphate
400 mM NaCl
500 mM imidazole
pH 8.0

We first prepared the column His-trap-FF 5 mL. Then, the machine (ÄKTA system, GE Healthcare Life Sciences) for purification was prepared.
Settings (UNICORN start 1.0) for sfGFP purification. Variable list.

The machine was then switched on and the affinity chromatography was conducted using UNICORN Control Software.

After this, 30 µL of protein sample was taken before dialysis to run it on a 12% gel (SDS-PAGE).
Dialysis was performed with 1L of 1xPBS buffer O/N at 4°C for 48 hours. We used water to wet the membrane tubing before starting the dialysis.

Day 5

We took 30 µL of our protein after dialysis for SDS-PAGE.
Concentrating the protein:

Vivaspin Protein Concentrator Spin Columns that we picked had molecular weight cut off 15 000 kDa since the size of our protein was 27 kDa.
The columns were centrifuged for 15 min, 4000 rpm, 4°C. There was precipitation.
Then we continued with the centrifugation until we had following final volumes:

Column nr. 1: 1 mL (4x15min)
Column nr. 2: 1.9 mL (5x15min)

Measurement of the protein concentration:

The protein was centrifuged at max speed, 5 min (each tube).
We made 50 µL aliquots into Eppendorf tubes. 30 µL of our protein was taken for running on the gel.
We had three different batches (1 from column nr. 1 and batch nr. 2 and nr. 3 from column nr. 2):

Batch nr. 1: 1 mL of protein solution, added 166ul of 60% glycerol
Batch nr. 2: 1 mL of protein solution, added 166ul of 60% glycerol
Batch nr. 3: 877 µl of protein solution, added 146ul of 60% glycerol

We did not have liquid nitrogen at that moment so we just froze the samples at -80ºC.

We used 6L of the running buffer.
Dialysis was done conducted.
Aliquots in the end were not made. The samples were stored in cold room at 4ºC in test tubes.
SDS-PAGE was not conducted either.