19/07/2017

Media Preparation:

LB agar Miller: 4 x 200 mL bottles

Add 8 g of agar in scott bottle.
Pour in 200 mL of distilled water.
Gently shake.
Autoclave at
Store at room temperature.

LB broth Miller:

2 x 25 g in 1 L.

5 x 50 mL in conical flasks.

50 x 10 ml in universal flasks.

24/07/17

Media Preparation (had to remake the Agar)

8x(200ml dH2O + 8g LB Agar).

Cultivating E.coli part 1

Pouring out agar plates..
Streaking plates with E.coli..
Put plates in incubation at 37’C for 15 hours max (put in at 6.08pm.).

Cultivating E.coli part 2 (Round 1)

Pick single colony of the cells from the LB agar plate into 10 ml of LB media containing no antibiotics OR specific for cell type..
Grew the cultures overnight at 37’C with shaking incubator at 250 rpm..

Pre pouring reagents for competent cells

0.1 M MgCl2 solution: 2.033g of MgCl2 6H2O in 100 ml H20 in a 100ml Pyrex Bottle..
0.1 M CaCl2 solution: 11g of Cacl2 in 500ml H20 in a 500ml Pyrex Bottle..
50% Glycerol solution: 10 ml of glycerol & 10ml H20 in 20ml culture tube with screw cup, autoclave at 4’C..

Competent cells (Round 1)

Inoculate 200 mL of pre-warmed to 37 degrees C LB medium (w/out antibiotics) with 10 mL of overnight E.coli cultures..
Incubate at 37 degrees C for 60 min with vigorous shaking 250 rpm or until OD600 is 0.4-0.5..
Put flask on ice for 30 min. At the same time chill sterile falcon tubes..
Aliquot culture into 50 mL each falcon tube (4x50).
Harvest cells by centrifugation for 7 min at 3,500 rpm at 4 degrees C and discard supernatant.
Resuspend cells with 12.5mL of MgCl2.
Centrifuge for 7 min at 3,500rpm at 4 degrees C and discard supernatant.
Resuspend cells with 25 mL of CaCl2 then incubate on ice for 30 min..
Centrifuge for 7min at 3,500rpm at 4 degrees C and discard supernatant.
Resuspend cells with 700 microL of CaCl2 and 300 microL of 50% glycerol. Final volume 1 mL..
Aliquot 50 microL aliquots into 1.5mL sterile eppendorf tubes and store at -80 degrees C..

Table of measured OD (during step 2):

Time (min)	OD600 batch 1	OD600 batch 2
0	0	0
15	0.043	0.047
30	0.031	0.012
45	0.065	0.005
60	0.108	/
75	0.150	/
90	0.422	/

NB: Calibrate with LB before inoculation, not with water.

Kanamycin plates

Made 23 kanamycin plates.

MgCl2

Made 200 mL of 0.1M MgCl2.

31/07/2017

Assaying Competency of E.coli competent cells (Round 1) - failed attempt of transformation

Serial dilute DNA given by Sarah: serial dilute 6 times to change concentration from micrograms/microL to picograms/microL - 45 microL of water + 5 microL of DNA.

Transform 100 microL competent cells with.

2 x 20 picograms of DNA (2 microL) 2 x 100 picograms of DNA (10 microL) 3.

Add 900 microL of LB media to all four .

4.

Incubate 60min at 37 degrees C.

5.

Plate 100 microL of 20 picograms and 100 picograms on LB+kanamycin plates.

Plate 800 microL of 20 picograms and 100 picograms on LB+kanamycin plates. 6.

Incubate plates at 37 degrees C overnight.

NB: Cells did not grow at all. Initially thought it was either 1) pb during transformation, or 2) cells are not competent. It turned out that we hadn’t even done a transformation procedure - the above protocol is a recap of transformation.

01/08/2017

LB Broth:

3 x 1L flasks.

Stock Solution of DNA

Use eppendorf tube labelled ‘1’ (first step of serial dilution carried out on 31/07).

Transfer 5 microL to next tube containing 45 microL of water (RNA-free).

Repeat 5 times until you reach tube 7 - at which concentration will be picograms per microliter..

Tube ‘1’ contained enough to make approx. 9 tubes with a concentration of picograms per microliter and volume of 50 microliters.

Cultivating E.coli part 2 (Round 2)

Inoculate 6 universal bottles containing LB broth with E.coli.

Incubate overnight in shaking incubator at 37 degrees.

02/08/17

Transformation (Round 1)

Transformation of E.coli competent cells made on 27/07/17 with Dr Sarah Coleman.

Pipette 5 Eppendorf tubes.

Take 50 ul of prepared E.coli competent cells and put on ice for 5 mins..

Add 1 ul of plasmid DNS and incubate on ice for 30 minutes..

Heat shock for 30 seconds at 42’C..

Put on ice for 2 minutes..

Add 900 ul of LB media..

Incubate for 1 hour at 37’C..

Plate on LB agar petri dish with appropriate antibiotic..

Incubate at 37’C overnight, (minimum 15 hours and maximum 24 hours.).

Tubes

1

2

3

4

5

Volume of DNA ul

2

10

2

10

N/A

Origin

DNA

IGEM

Sarah

NOTE: No growth at all: 1) the cells were not competent, 2) PLates have too much antibiotic or wrong antibiotic.

Biofilm culture part 1 (Round 1)

Inoculate Pseudomonas putida in 3-to-5-ml culture and incubate at 30/37 degrees overnight..

27/07/17.

Competent cell making (Round 2)

Competent cells are made again following the same procedure as.

03/08/17

Biofilm culture part 2 (Round 1)

Dilute culture 1:100 in LB broth (1 mL culture + 100 mL broth). .

Pipet 100 μl of diluted culture into each of four wells in a fresh microtiter plate which has not been tissue culture treated. .

Cover plate and incubate at 30/37 degrees 24 hours/48 hours..

We are going to make 2 plates - 1 round bottomed and 1 flat bottomed. Reason being that round-bottomed might not work/incompatible with plate reader but if it does, uni has plenty of them that no one uses.

Transformation (Round 2)

Protocol same as 31/07/17.

04/08/17

Biofilm culture part 3 (Round 1)

Tried to continue with biofilm culture but did not find person in time to help us work out spectrometer (Henry Song)..

07/08/17

Biofilm Culture part 3 (Round 1)

Set up four small trays in a series and add 1 to 2 inches of tap water to the last three. The first tray is used to collect waste, while the others are used to wash the assay plates..

Remove planktonic bacteria from each microtiter dish by briskly shaking the dish out over the waste tray. To wash wells, submerge plate in the first water tray and then vigorously shake out the liquid over the waste tray. Replace water when it becomes cloudy..

Add 125 μl of 0.1% crystal violet solution to each well. Stain 10 min at room temperature..

Shake each microtiter dish out over the waste tray to remove the crystal violet solution. Wash dishes successively in each of the next two water trays, and shake out as much liquid as possible after each wash. .

Invert each microtiter dish and vigorously tap on paper towels to remove any excess liquid. Allow plates to air-dry. At this stage, the staining is stable and the dried plates may be stored at room temperature for at least several weeks..

Add 200 μl of 30% acetic acid (or another appropriate solvent) to each stained well. Allow dye to solubilize by covering plates and incubating 10 to 15 min at room temperature. .

Briefly mix the contents of each well by pipetting, and then transfer 125 μl of the crystal violet/acetic acid solution from each well to a separate well in an optically clear flat-bottom 96-well plate. Measure the optical density (OD) of each of these 125-μl samples at a wave-length of 500 to 600 nm. .

Diluted 60% acetic acid to get 30% acetic acid: 30mL acetic acid + 30mL water.

OD readings of flat-bottom plate:

Biofilm Culture part 1 (Round 2)

Inoculate Pseudomonas putida in 3-to-5-ml culture and incubate at 30/37 degrees overnight.

Transformation (Round 3):

09/08/17
To assay biolfilms - best use ‘endpoint’ setting in spectrometer. It would seem that the best time for CV incubation is 10 min. Both dyes present fairly similar values however, the mixed acetic acid/CV solution has highest OD.

Team