Team:Paris Bettencourt/Notebook/Phage Sensor/Friday 9th August.html


Phage Sensor

Friday 9th August

Liquid culture, Gel, Trafo of BBa_E1010 (pSB1C3 backbone) into NEB turbo, Glycerol Stock, Making electro competent cells of pir+ E.coli, Transforming pKD13 into pir+ E.coli, Miniprep NEB turbo with BBa-J04450:, Liquid culture and Patch plates.

Liquid culture
1:1000 from liquid: FR-433 (M13), 10 ml
pir+ E.coli: from plate, 25 ml
XL10: from plate 10 ml

Gel: rest of circular backbone PCR product, 1% Agar, 100V, 20 min
SPCR4-7-8-9-10 (40.5) SPCR4-7-8-9-10 (60.5)
Gel picture:
didn’t work

Trafo of BBa_E1010 (pSB1C3 backbone) into NEB turbo
1) Thaw competent cells on ice.
2) Place 20 µl of cells in a pre-chilled Eppendorf tube.
3) Add 2.5 µl of plasmid (from Biobrick stock)
4) Mix gently by flicking the tube.
5) Chill on ice for 10 minutes.
4) Heat shock at 42 °C for 30 seconds
5) Return to ice for 2 minutes.
6) Add 200 µl LB medium and recover the cells by shaking at 37 °C.
Chloramphenicol: 60-120 minutes
7) Plate out the cells on selective LB (10ul)
8) Incubate at 37 °C. Transformants should appear within 12 hrs.

Glycerol Stock FR-433 (M13) -> sSP012
Glycerol Stock pir+ -> sSP013
Glycerol Stock pKD13 -> sSP014
Glycerol Stock NEB turbo with BBa-J04450 -> sSP015
Take 1.5 ml of ON culture + 500ul Glycerol (60%)
Freeze at -80°C

Making electro competent cells of pir+ E.coli
Preparation of Electrocompetent Cells
Note: Competent cells should never be vortexed, as this will cause them to lyse and release salts into the media. Resuspend cells by pipetting up and down with a large pasteur pipet. Once they are chilled, cells should be continuously cold.

1) The night before the transformation, start an overnight culture of cells.
pir+ E.coli
2) The day of the transformation, dilute the cells 100X.
100 ml LB
Grow at 30°C for about 90 minutes.
3) When the cells reach an OD600 of 0.2.
4) Harvest the cells.
When the cells reach an OD600 of between 0.6 and 0.8.
Split the culture into 2x 50 ml falcon tubes, on ice.
Centrifuge at 4 °C for 10 min at 4000 rpm.
5) Wash and combine the cells.
Remove the supernatant.
Resuspend the cells in 2x 25 ml of ice cold water.
Combine the volumes in a single 50 ml falcon tube.
6) Wash the cells 2 more times.
Centrifuge at 4 °C for 10 min at 4000 rpm.
Resuspend in 50 ml of ice cold water.
7) Wash and concentrate the cells for electroporation.
Centrifuge at 4 °C for 10 min at 4000 rpm.
Resuspend in 1-2 ml of ice cold water.
We will use 200 µl of washed cells per transformation.

Transforming pKD13 into pir+ E.coli
1) Prepare BD tubes with a pipette filled with LB at the interior of each tube (pipette supplied with the electroporation cuvette)

2) Test the purity of the electrocompetent cells.
Add 200 ul of washed cells to a cuvette.

3) Mix the cells and DNA in a cuvette.
200 ul of washed cells with 200 ng of PCR product.
Keep the cuvette on ice until just before the electroporation.

4) Preload a pipette with 1 ml of LB.

5) Pulse the cuvette with voltage.
Dry the electrodes with a kimwipe prior to loading.
Use the EC2 setting.

6) Listen for arcing.
A cracking sound means all the cells are dead.
Note the time constant: 5 is good, 5.8 is great.

7) Immediately recover the cells.
Add the 1 ml of preloaded LB and pipet up and down to mix.
Collect 1 ml of cells, some volume is lost in the cuvette.

8) Incubate 2 h at 37 °C with shaking.

9) Plate 100 ul of recovered cells on selective plates.
Use antibiotic appropriate to the part being integrated.
Let the other 900 ul rest overnight at room temperature.

10) Concentrate and plate the remaining cells
Spin down quickly and resuspend in 100 ul LB before plating.

Transformed cells should be incubated at 37 °C.
Colonies should appear 24-48 h after plating.

Miniprep NEB turbo with BBa-J04450:
1) Pellet 2x 9 ml of liquid culture (4000rpm, 10 min)
2) Discard supernatant
3) resuspend the cells in 250 µl of resuspension solution
4) add 250 µl of lysis solution, mix by inverting 4-6 times
5) add 350 µl of neutralization solution
4) centrifuge for 5 min
5) transfer supernatant to spin column
6) centrifuge for 1 min
7) discard flow through
8) add 500 µl wash solution and centrifuge for 1 min , discard flow through(repeat this step)
9) centrifuge for 1 min to remove left over liquid
10) transfer the column on a 1.5 ml tube
11) add 50ul of elution buffer and incubate for 2 min
12) centrifuge for 2 min
13) Nanodrop the concentration and freeze at -20°

J04450: 0ng/ul

Liquid culture of: sSP012
XL-10 = sSP016
sSP017= Litmus

Patch plates of: