Team:Dundee/Notebook

Notebook

Protocols

Chemically Competent E. coli Cells

1. Dilute overnight culture of E. coli cells 1/100 into 5 ml of fresh LB.
2. Incubate cells at 37°C with shaking until an OD₆₀₀ of 0.5-0.8 is reached.
3. Chill the cells on ice for 20 min before harvesting at 4000 rpm at 4°C for 10 min.
4. Resuspend pellet in 0.5 ml ice cold transformation buffer (TB) and leave on ice for 10 min.
5. Divide cells into 100 µl aliquots, snap freeze in liquid nitrogen and store at -80^oC.

Transformation of chemically competent E. coli cells

1. Thaw 100 µl of competent cells on ice for 20 min and mix with either 1 µl of plasmid DNA or 10 µl ligation reaction.
2. Chill cells on ice for 20 min.
3. Heat shock cells at 42°C for 90 seconds, before chilling cells on ice for 2 min.
4. Add 1 ml of LB to cells and recover at 37°C with shaking for 1 hour.
5. Pellet cells by centrifugation at 13 000 rpm for 3 min.
6. Remove supernatant resuspend in 200 µl of LB and plate onto LB medium containing appropriate antibiotics.
7. Incubate plates overnight at 37^oC.

Polymerase Chain Reaction

1. PCR reaction mix set up as follows (final volume = 50 µl)

2. Thermocycler protocol (Elongation time was altered according to desired length of PCR product).

3. Run products on a 1% agarose gel for analysis.

Agarose Gel Electrophoresis

DNA products are run on a 1% (w/v) agarose gel to separate charged nucleic acids by size for analysis or purification. GelRed used to stain nucleic acids and gels visualised on a BioRad GelDoc.

For 1% (w/v) agarose gel

1. Weigh out 1g of agarose and pour into 100 ml of 1 x tris-acetate-EDTA (TAE) buffer.
2. Heat agarose solution in the microwave until the agarose had dissolved (1-3minutes) and there is a rolling boil.
3. All the solution to cool before Gel Red (6µl per 100ml of 1% (w/v) agarose solution) is added.
4. Pour agarose solution into a gel tray with the required combs in place.
5. Allow the agarose solution to cool in the gel tray before loading the DNA into the gel.
6. Mix the DNA samples with 10 x DNA loading dye and load on the gel with the DNA marker (Roche).
7. Run the gels in 1 x TAE buffer for 30 min at 100 V. After sufficient separation the gel is visualised under UV light on the BioRad GelDoc.

TAE buffer recipe (pH 7.6): 40mM Tris, 20mM acetic acid, 1mM EDTA

QIAquick Gel Extraction Kit protocol (Qiagen)

QIAquick Gel Extraction Kit Protocol from Qiagen was used. This was designed to extract and purify DNA from agarose gels in TAE buffer.

1. Excise DNA from agarose gel with a clean sharp scalpel and add into a tube.
2. Add 800 µl of buffer QG of gel slice.
3. Incubate at 50°C for 10 min or until gel slice has completely dissolved. To help dissolve gel, mix by vortexing the tube every 2-3 minutes.
4. After gel slice has dissolved completely the colour of the mixture should be yellow.
5. Add 200 µl of isopropanol to sample and mix.
6. Add 500µl of buffer QG to the QIAquick column and centrifuge for 1 minute. Discard flow-through and place the QIAquick column back into the same tube.
7. To wash, add 750 µl buffer PE to QIAquick column and centrifuge for 1 minute. Discard flow-through and place the QIAquick column back into the same tube. Centrifuge the QIAquick column in the 2ml collection tube provided for 1 minute to remove residual wash buffer.
8. Place the QIAquick into a clean 1.5 ml microcentrifuge tube
9. To elute DNA, add 50 µl of water to the centre of the QIAquick membrane and centrifuge for 1 minute.
10. To analyse purified DNA of a gel, add 1 volume of loading dye to 5 volumes of purified DNA.

Restriction digest

Restriction digests are performed to prepare DNA for ligation. Enzymes used were supplied by Roche and New England Biolabs. 60 µl reactions following clean up after PCR were set up.

1. 60 µl reactions following clean up after PCR were set up as follows:
• DNA = 50 µl
• Buffer = 6 µl cognate digestion buffer, for enzyme (s)
• Restriction enzyme 1 = 1.5 µl
• Restriction enzyme 2 = 1.5 µl
• H20 = 1 µl
2. Incubate at 37^oC for 3 hours.
3. If digesting a vector, add 2.5 µl of alkaline phosphatase at 2 hours, and then add a second aliquot at 2.5 hours.
4. Digests can then be purified using the Qiagen PCR purification kit.

QIAquick PCR purification kit protocol (Qiagen)

1. Add 5 volumes of buffer PB to 1 volume of PCR reaction.
2. Put sample in QIAquick column, centrifuge for 1 min at 13 000 rpm and discard flow-through.
3. Add 750 µl of buffer PE to the column and centrifuge for 1 min, discard flow through.
4. Centrifuge again for 1 min to remove residual buffer.
5. Place column in clean Eppendorf.
6. To elute DNA, add 50 µl of water to the centre of the QIAquick membrane and centrifuge for 1 min.

Polymerase Chain Reaction

1. Ligation reactions set up as follows:

2. Incubated at room temperature for 3 hours and then transform into chemically competent E. coli cells.

Plasmid DNA Miniprep

Plasmids were isolated using a QIAprep Spin Miniprep Kit protocol (Qiagen).

1. Spin down overnight culture of cells for 5 minutes at 4000 rpm and discard supernatant.
2. Resuspend the resulting pellet in 250 µl of buffer P1 (with RNase A added) and transfer to a clean eppendorf.
3. Add 250 µl of buffer P2 and mix.
4. Add 350µ l of buffer N3 and mix.
5. Centrifuge samples for 10 min and 13 000 rpm in table top centrifuge.
6. Apply the supernatant to QIAprep spin column by decanting or pipetting and centrifuge at 13 000 rpm for 1 min and discard the flow-through.
7. Wash QIAprep spin column in 500 µl of buffer PB, centrifuge at 13 000 rpm for 1 min and discard flow through (optional).
8. Wash spin column with 750 µl buffer PE and centrifuge at 13 000 rpm for 1 min and discard flow through.
9. Centrifuge at 13 000 rpm for 1 min to remove residual buffer.
10. Place column in clean Eppendorf and elute with 50 µl of water, let sit for 3 min, centrifuge for 1 min at 13 000 rpm.

Colony PCR

Colony PCR was used to identify the desired clones on a plate of transformed cells. PCR was performed on samples using Taq polymerase supplied by Aligent Technologies.

A single colony was picked and re- suspended in 30 μl of ddH2O. The cells were boiled for 10 min and then centrifuged for 1 min at 13 000 rpm in a microfuge. 5 μl of DNA was then used as part of the reaction mixture outlined below.

Glycerol Stock

1. Pick single colony from agar plate.
2. Inoculate 5 ml LB broth overnight (with appropriate antibiotic).
3. Add 1 ml of overnight culture to 1 ml of 60% glycerol in a cryotube (1:1 ratio).
4. Freeze at -80°C.

SDS – PAGE and Western Blot analysis

Protein samples were separated on resolving gels containing 12% acrylamide, and a stacking gel of 6% acrylamide, alongside 5 μl of protein ladder (Precision plus Protein All Blue Standard, Bio-Rad).

For 12% acrylamide gel:

Resolving gel recipe:

After casting the resolving gel, add a layer of isopropanol on top to get rid of any bubbles and keep a straight line on the top. Once the resolving gel is set, the isopropanol can be poured off then the stacking gel added.

Stacking gel recipe:

After adding the stacking gel an appropriate comb was put in place and left to set. Samples ran as follows:

1. Samples separated in 1 x SDS running buffer at 80 V until samples reached the resolving gel. The voltage was then increased to 180 V until the dye front reached the bottom of the gel.
2. Proteins were then transferred onto nitrocellulose membrane by semi-dry transfer. This was performed at 5V for 40 min. Place gel between 2 x Whatmann filter paper, nitrocellulose membrane, 2 x Whatmann filter paper all soaked in transfer buffer.
3. Membrane blocked overnight in 5% semi-skimmed milk dissolved in TBST (Tris-buffered saline, 0.1% Tween20) at 4^oC.
4. Blocking buffer then removed and the primary antibody added to fresh 5% milk in TBST for 1 hour at room temperature.
5. This was followed by four 15 min wash steps with TBST.
6. The membrane was then incubated with the secondary antibody (unless primary antibody is conjugated to horseradish peroxidase-HRP-conjugaed) in 5% milk in TBST and incubated at room temperature for 1 hour.
7. The membrane was again washed four times for 15 min with TBST at room temperature.
8. Photo-detection of the cross-reacting bands used enhanced chemiluminescence (ECL)reagent (luminol+HRP) and exposure using the GENEGnome.
9. The following antibody dilutions were used: -GFP 1:2000,  -HA-HRP 1:5000.

Transfer buffer recipe:

• 0.6g Tris
• 2.9 g glycine
• 20 ml methanol
• 180 ml H20

Subculture of to induce Toxic Expression

To test the toxicity of our toxic chimeras which were cloned into pBAD, we needed to induce the pBAD inducible promoter with arabinose in order to get toxic production.

1. Overnights of the colonies containing the toxin were taken
2. 250μl of these cells were added to 3 conical flasks containing 25ml of LB and 25μl of kanamycin antibiotic to select for the toxins in the pBAD vector.
3. The conical flasks were then incubated at 37°C for 1.5 hours, until they reached an OD of between 0.5-0.8A.
4. In one flask glucose was added to a final concentration of 0.2% glucose, in another final concentration of 0.2% arabinose, and in the 3rd flask 0.5% arabinose.
5. They were then incubated again at 37°C, and the OD was measured and samples were taken roughly every 40 minutes. The final samples taken were used for western blot.

Preparing samples for western blot

1. 1ml of each sample was spun down in a table top centrifuge for 1 minute.
2. The pellet was then resuspended in 100μl of laemmeli sample buffer with β-mercaptoethanol (50μl β-mercaptoethanol in 950μl laemmeli).
3. The samples were then boiled at 95°C for 10 minutes.
4. Load between 5-15μl of samples into acrylamide protein gel.

TCA precipitation of supernatant samples

1. Supernatant samples were filter sterilised and made up to 1ml with LB.
2. Add 5μl deoxycholate (10mg/ml) and 110μl Trichloroacetic acid (TCA) (1g/ml).
3. Samples were mixed thoroughly and kept on ice in cold room for a minimum of 3 hours.
4. They were then centrifuged at 4°C at 11,000g, with the lids of the Eppendorf all facing inwards.
5. Remove supernatant (put TCA in TCA waste)
6. 500μl of 80% ethanol were pipetted on inside of Eppendorf, without disturbing the pellet, to wash.
7. Spin in centrifuge for 5 minutes and remove supernatant.
8. Dry pellet by leaving them on 37°C heat block with lids open for 20 mins.
9. Pellet then resuspended in 50μl Tris-SDS.
10. 50μl of laemmeli sample buffer with β-mercaptoethanol (50μl β-mercaptoethanol in 950μl laemmeli) was then added and samples were boiled at 95°C for 10 minutes.

Preparation of promoter-GFP samples for western blotting

1. The OD₆₀₀ of the over night incubated promoter constructs was measured (450µl fresh LB and 50µl of the over night culture per cuvette; 500µl fresh LB as a Blank; then calculate actual OD₆₀₀)
2. The cultures were pelleted 13000 rpm for 7 minutes
3. Normalised to an OD₆₀₀ of 1 with Lamelli buffer
4. 15µl from the Lamelli buffered samples was loaded into each well of the 12% Acrylamide protein gel
5. Follow procedure for SDS-PAGE and Western Blot Analysis

Measurement of GFP and OD₆₀₀ using Plate Reader

Measurement of GFP fluorescence from our pH promoter – gfp constructs.

1. Overnight cultures harbouring the pSB1C3-Pasr-gfp, P-gadA-gfp, PacrRA-gfp were diluted 1/10 into 1 x MOPS buffered LB (to various pH conditions with either concentration H2SO4 or NaOH).
2. The adjusted pH values were pH 2, 4, 5, 6, 7 and 8. The 1 x MOPS buffered LB was then filter sterilised.
3. 3. 200 µl loaded into 96 well plate and the OD₆₀₀ as well as GFP fluorescence was measured (ex:em) over 20 hour every 20 min.

Measurement of GFP fluorescence from our bile salt promoter – gfp construct.

1. Overnight cultures harbouring the pSB1C3- PacrRA-gfp construct samples (for details on which were used view ‘LETTER CODE’ below table) were normalised to an OD of 1 with minimal media
2. 6µl of the over night normalised cultured were transferred into each well from row A to D column 1 to 12.
3. A 1mM stock of Sodium Cholate – in sterile water was diluted to concentrations of 5µg/ml, 10µg/ml and 25µg/ml with minimal media and the normalised cultures. (the final concentration in well columns 1-3 was 25µg/ml in 200µl. The 200µl includes the 6µl from the over night normalised samples.
4. OD₆₀₀ as well as GFP fluorescence was measured (ex:em) over 20 hour every 20 mins.

GFP Fluorescence Detection Using the Microscope:

This protocol was used to measure the GFP fluorescence of our bile salt promoter construct PacrRA-gfp.

1. Competent MG1655 cells were transformed with pUniprom-RamA and PacrRA-gfp. RamA acts as the transcriptional activator. In order to investigate the effect of RamA, sufficicent control transformations were also set up.
2. The standard transformation protocol was followed and cells finally plated onto LB agar or MacConkey containing 2 x Amp/ 2x Cml.
3. Transformations incubated overnight at 37^oC.
4. GFP fluorescence was viewed with the fluorescence microscope (for quantitative measurements the plate reader was used)

E. coli Toxicity Assay

Plate toxicity experiments were carried out to test the toxicity of our synthetic toxins. Expression of our synthetic colicins in E. coli MG1655 was under the control of the L-arabinose-inducible, D-glucose-repressible pBAD promoter on vector pBAD18-Kn.

1. Plasmids required for the experiment were transformed into MG1655 chemically competent cells and plated onto LB containing 0.2% D-glucose and incubated at 37^oC overnight.
2. The following day colonies were picked and re-suspended in LB to a calculated OD₆₀₀ of 1.0.
3. Serial dilutions 1:10 to 10-6 were prepared and 5 µl of each dilution spotted onto LB plated containing 0.2% D-glucose, and increasing concentrations of L-arabinose (0.02%, 0.2%, 0.4%).
4. The plates were then incubated overnight at 37^oC.

E. coli Toxicity Assay

We were unable to see any toxicity from our synthetic toxins when they were overexpressed in E. coli . This may be due to the fact that our warheads on our synthetic colicins degrade the peptidoglycan wall and if the toxin is not secreted it will not reach its target. We were unable to see any secretion of our synthetic toxins and so we decided to lyse our cells and release our toxins and test for toxicity against E. coli . The overexpression of our synthetic toxins protocol was followed and then lysis as follows:

1. 25 ml of 0D₆₀₀ = 2.0 of MG1655 cells harbouring pBAD18-Kn with our synthetic toxins were pelleted by centrifugation at 4000rpm.
2. The pellets were resuspended in 1.5 ml of 1 x PBS.
3. The cells were lysed for 1.5 min using a sonicator (20% amplitude, 3 x 30sec).
4. MG1655 E. coli cells were plated onto LB agar plates using a cotton bud (this acts as our target for our synthetic colicins).
5. Lysed MG1655 cells with our pBAD18-Kn synthetic toxin constructs were then spun down to remove any cell debris.
6. 5 µl of cell lysate was spotted onto the plate of MG1655 E. coli cells.
7. Positive and negative controls were also spotted alongside our synthetic toxins.
8. Spots were dried by a flame and then incubated overnight at 37°C.

Lab Book - Holly

Week 1

1. Familiarisation with methods and techniques

2. Restriction digest of AcrRA Bile Salt promoter

• Digested with Spe1 and Pst1
• Run on a gel

22/6/16

1. Restriction digest of Asr and GadA pH sensitive promoters

• Digested with Spe1 and Pst1
• Total volume = 50μl
• Alkaline phosphatased
• Ran on gel then gel extracted
• Nanodrop concentrations

o GadA = 22.9ng/μl

o Asr = 21.0ng/μl

2. Restriction digest of RamA

• Digested with EcoR1 and Pst1
• Total volume = 30μl
• Ran on a gel

3. Ligation of RamA with pSB1C3 vector cut with EcoR1 and Pst1

• Ligated overnight

Week 2

1. Restriction digest RamA in pSB1C3

• Digested with Xba1 and Pst1
• Total volume = 30μl
• Nanodrop concentration = 13.1ng/μl

2. Restriction Digest GFP

• Restriction digest with Xba1 and Pst1
• Total volume = 50μl
• Nanodrop concentration: 9.7 ng/ul

3. Ligation of RamA with RBS vector

• Included vector only control, 2:1 and 3:1 reaction
• Ligated overnight

28/6/16

1. Restriction digest of Colicin E3 immunity

• 2 reactions

o Digested with EcoR1 and Pst1

o Digested with Xba1 and Pst1

• Nanodrop concentrations

o EP digest = 12.7ng/μl

o XP digest = 5.2ng/μl

2. Ligation of pSB1C3 RBS with E3 immunity (cut XP)

• Included vector only control, 2:1 and 3:1 reactions
• ligated for 3 hours
• Transformed into JM110

29/6/16

1. Miniprep of pSB1C3

• Nanodrop concentration

o 72.2ng/μl

o 102.1ng/μl

2. Restriction digest of pSB1C3

• Digested with EcoR1 and Pst1
• Total volume = 50μl
• Alkaline phosphatased
• Run on gel

30/6/16

1. Miniprep E3/RBS overnight

2. PCR of Colicin E9 and iA immunity gblocks with primers

• Dry gblocks resuspended in 50μl of water
• Primers made up with water the diluted 1/10
• Ran on gel

1/7/16

1. Restriction digest of Colicin iA and E9 immunities

• Digested with EcoR1 and Pst1

Total volume = 30μl

Week 3 + Week 4

1. Restriction digest RBS-GFP

• Xbal-Pst
• Nano drop GFP = 9.7 ng/ul

2. Colony PCR acrRA

• Run on gel

5/7/16

1. Ligations with GFP

• acrRA- GFP ligation
• gadA-GFP ligation
• asr-GFP ligation

2. Transformations into JM110 cells

7/7/16

1. Colony PCR of ligations from the 05/07

2. Over night cultures of promoter-GFP constructs from transformations

8/7/16

1. Miniprepped the over nights:

• Nanodrop concentrations:

o gadA-GFP colony 1: 115.2 ng/ul

o gadA-GFP colony 2: 256.3 ng/ul

o gadA-GFP colony 3: 226.2 ng/ul

o gadA-GFP colony 4: 172.5 ng/ul

o asr-GFP colony 1: 170.4 ng/ul

o asr-GFP colony 2: 134.4 ng/ul

o asr-GFP colony 3: 149.7 ng/ul

o asr-GFP colony 4: 127.9 ng/ul

2. Samples prepared for sequencing with forward and reverse primers

3. Restriction digest for promoters asr and gadA

• Cut with Xbal-Pst
• Alkaline phosphetased

9/7/16:

1. Transformation of acrRA from kit plate

• Suspend part from kit plate in 50ul H₂O (sterile) and keep as stock
• Transform into JM110 cells

2. Run gel of restriction digests of asr and gadA

3. Over nights of asr and gadA

10/7/16

1. Miniprep over nights of asr and gadA

2. Over night cultures of acrRA transformations

Week 5

1. Miniprep acrRA over night cultures

2. Restriction digest acrRA with Xbal-Pst

3. Run digest on gel.

4. Ligation of acrRA with GFP

5. Transformation of ligation into JM110 cells

6. Over night of gadA-GFP and asr-GFP and acrRA-GFP

12/7/16

1. Mini-prep over nights:

• Asr-GFP
• gadA-GFP
• acrRA-GFP

2. Send samples from above table for sequencing

3. Transformations:

· gadA-GFP à MG1655 cells

· asr-GFP à MG1655 cells

· acrRA-GFP à MG1655 cells

14/7/16

1. Preparation of MOPS buffered LB

• 900ml 10x MOPS to 10ml LB
• Adjust pH with concentrated HCl or NaOH

· Filter sterilise - ready for use.

2. Over night cultures from JM110 plates ready for glycerol stocking

15/7/16

1. Glycerol stocking of promoter constructs with GFP

2. Spin down remaining over night culture and freeze pellet ready to mini prep (16/7/16)

3. Made 12% acrylamide protein gel

4. SDS PAGE of asr and gadA

• Pellet 1ml of over night culture
• Measure OD
• Adjust to OD of 1 with Lamelli buffer
• Insert 10ul into each well of the gel

16/7/16:

1. Mini prep samples from over night

• acrRA: 115.9 ng/ul
• gadA: 84.8 ng/ul
• asr: 64.0 ng/ul
• gadA-GFP: 145.7 ng/ul
• asr-GFP: 144.6 ng/ul

Week 6

1. Over night cultures

• Asr
• Asr-GFP
• gadA
• gadA-GFP

18/7/16

1. Run plate reader experiment

• 96 well plate
• Measure OD₆₀₀
• Measure GFP fluorescence
• 20h

19/7/16

1. Collect data and analyse

20/7/16

1. Restriction Digest of ramA

• BamHI and SalI

2. Ligation of ramA with pUniprom

3. Transformation of ligation into MG1655 cells

Week 7

1. Repeat of ligations (ramA into pUniprom)

2. Repeat of transformations into MG1655 cells (ramA-pUniprom)

3. Transformation of empty pUniprom in MG1655 cells

27/7/14

1. Make MG1655 competent cells

2. Over night:

• pUniprom empty (MG1655 cells)
• acrRA-GFP (psb1c3, MG1655 cells)

28/7/16

1. Glycerol stock parts

• acrRA-GFP

2. Double transform ramA on pUniprom with acrRA-GFP on psb1c3 into MG1655 cells

29/7/16

1. Re-make MOPS buffered LB, as last time to have it fresh and sterile

2. Over night

• gadA
• gadA-GFP
• asr
• asr-GFP

30/ 7/16

1. Repeat of plate reader:

• 96 well plate
• Measure OD₆₀₀
• Measure GFP fluorescence
• 20h

Week 8

1. Repeat double transformations in MG1655 cells on nutrient agar

• acrRA-GFP(psb1c3) + ramA(pUniprom)
• ramA(pUniprom) + psb1c3 empty
• acrRA-GFP + pUniprom empty
• empty pUniprom + empty psb1c3

2/8/16

1. Streak plate from nutrient agar plates onto MacConkey agar plates

• acrRA-GFP + ramA
• ramA
• acrRA-GFP
• psb1c3 empty and pUniprom empty

3/8/16

1. Make protein gels (12% acrylamide) x 2

2. View MacConkey plates and Nutrient agar plates under fluorescence microscope and image

4/8/16

1. Over night anaerobic 5ml of:

• acrRA-GFP + ramA
• acrRA-GFP
• ramA
• psb1c3 empty and pUniprom empty

5/7/16

1. Over nights did not grow

2. Analyse data from plate reader done on 30/7/16

8/8/16

1. Make up MacConkey broth

· Add suggested amount of sterile water (see product description on container)

• Autoclave
• Use 5ml and antibiotocs for over night cultures

2. Over night cultures

· acrRA-GFP, ramA MacConkey broth

• acrRA-GFP MacConkey broth
• ramA MacConkey broth
• empty psb1c3 and empty pUniprom MacConkey broth

· acrRA-GFP, ramA nutrient broth

• acrRA-GFP nutrient broth
• ramA nutrient broth
• empty psb1c3 and empty pUniprom nutrient broth

3. Make up 12% acrylamide protein gels

9/8/16

1. Measure OD of over nights

2. Pellet over nights

3. Adjust OD to 1 wth Lamelli buffer

4. Run SDS PAGE (12% acrylamide) x2

10/8/16

1. Western blot

• Anti-GFP antibody
• Anti-HA antibody

- No Anti-HA tag picked up for ramA

11/8/16

1. Repeat SDS PAGE from 9/7/16 (12%acrylamide)

• Anti-HA antibody

- Still no results

Week 9

1. Make up stock of Sodium Cholate (1 mmol)

2. Over night

· acrRA-GFP, ramA nutrient broth

• acrRA-GFP nutrient broth
• ramA nutrient broth
• empty psb1c3 and empty pUniprom nutrient broth

17/8/16

1. Set up plate reader experiment for acrRA

18/8/16:

1. Analyse data from plate reader

19/8/7:

1. Restriction digest Chi……with …….

2. Ligate with acrRA promoter

3. Transform into MG1655 cells

Week 10

1. Transform ….into …. Cells

2. Over nights

23/8/16

1. Make protein gel (12% acrylamide)

2. Measure OD of over nights

3. Adjust to OD1 with lamelli buffer

4. SDS PAGE

• 10 ul into each well

24/8/16

1. Western blot of SDS PAGE from 23/8/16

• Anti HA antibody

25/8/16

1. Data analysis of plate reader experiments

26/8/16

1. Stomach Masher

Lab Book - Kieron

6/06/16

1. Prepare competent cells.

• DH5α and JM110 strains used.

· Stored in -80 degree Celsius freezer in 100ul aliquots for transformations.

2. Transform empty pSB1C3 plasmid with DH5α cells.

• 100ul cells used.
• 1ul plasmid transformed.

7/06/16

1. Prepare agarose gel.

• 300 ml TAE and 3g agarose used.
• 15ml gel red used.

2. Practice overnight culture technique.

• 5ml LB + 5ul antibiotic.

3. Miniprep transformed pSB1C3 in DH5α.

• Nanodrop concentration

o 142.69 ng/ul

8/06/16

1. Take Asr and GadA pH sensitive promoters out of distribution kit.

• Eluted with 10ul water.
• Frozen in Eppendorf.

9/06/16

1. Transform Asr and GadA promoters.

• DH5α cells used.
• 2ul transformed.

10/06/16

1. Miniprep transformed Asr and GadA.

• Nanodrop concentrations.

o 67.4 and 78.5 respectively (ng/ul).

27/6/16

1. Restriction digest RamA in pSB1C3 .

• Digested with Xba1 and Pst1.
• Total volume = 30μl.
• Nanodrop concentration = 13.1ng/μl.

2. Digest GFP.

• Restriction digest with Xba1 and Pst1.
• Total volume = 50μl.

3. Ligation of RamA with RBS vector.

· Included vector only control, 2:1 and 3:1 reaction.

• Ligated overnight.

4/7/16

1. Digestion of Colicin IA and E9 immunity proteins.

• Two digestions;

o Digested with EcoR1 and Pst1.

o Digested with Xba1 and Pst1.

• Total volume = 30μl.
• Run on Gel.

2. Test JM11O competent cells.

· Plate on chloramphenicol, ampicillin and plain agar.

3. Ligation of E9 and Ia immunities with pSB1C3 and RBS vector.

• Vector only, 2:1 and 3:1.

5/07/16

1. Digest RBS backbone and pSB1C3 backbone for stock.

• RBS cut Spe and Pst, pSB1C3 cut EcoR1 Pst.
• Both treated with alkaline phosphatase.
• Total volume 30ul.

2. PCR clean up digested backbones.

• Eluted with 30ul water.

6/07/16

1. Digest E9 and Ia immunities hat have been ligated with RBs backbone.

• Cut EcoR1, Pst.
• Total volume 30ul.
• Gel Run.

7/07/16

1. Send E9 and Ia samples for sequencing.

8/07/16

1. Re-miniprep E9 immunity, Ia Immunity and RBS backbone.

11/07/16

1. Re-digest Colicin E9 immunity protein.

• Gel Run

12/07/16

1. Digest full length colicins.

· Two reactions, one set cut EcoR1 and Pst, one set cut XbaI and Pst.

• Total volume 30ul.
• Gel Run

13/07/16

1. Ligate fill length colicins with pSB1C3 and RBS.

• EcoR1 and Pst cut ligated with pSB1C3.
• XbaI and Pst cut ligated with RBS.
• Vector only, 2:1 and 3:1.

14/07/16

1. Re-digest RBS Vector.

• Digested Spe1 and Pst1.

2. Transform Ligations of Full length colicins in pSB1C3 and RBS.

· 100ul JM11O cells and whole ligation mixture used.

15/07/16

1. Send RBS Vector for sequencing.

2. Miniprep transformations of full length colicins.

3. Send minipreps for sequencing.

18/07/16

1. Re-ligate Full length colicins with pSB1C3 ( primers ordered for RBS as the sequence is off)

2. Transform ligated colicin and pSB1C3.

· 100ul JM11O cells and whole ligation mixture used.

19/07/16

1. Colony PCR of transformed full length colicins.

• Gel Run.

20/07/16

1. More colonies from the full length colicin plates were overnighted.

21/07/16

1. Miniprep of overnighted full length coicins.

2. Digestion of miniprep

• Digested EcoR1 and Pst.

25/07/17

1. Ligate colicin E3, E9 and Ia immunity proteins with the cut promoters (GadA and AcrrA).

2. Transform immunity and promoter ligations.

26/07/16

1. Overnight transformations with immunities and promoters.

27/07/16

1. Miniprep overnight cultures.

2. Digest miniprep reactions.

• Digested EcoR1 and Pst.
• Run gel.

28/07/16

1. PCR amplify full length colicin Ia and RBS forward primer and HA tag reverse primer.

• Run gel.

1/08/16

1. Digest vector (immunity protein and promoter)

• Cut Spe and Pst.
• Treat with alkaline phosphatase.

2. Digest full length colicin.

• Cut XbaI and Pst.

2/08/16

1. Ligate digested vector and full length colicin.

2. Transform ligation reaction in JM110.

3/08/16

1. Carry out colony PCR of transformed vector and full length colicin.

• Run gel.

4/08/16

1. Re-ligate vector and full length colicin.

2. Transform reaction in JM110.

5/08/16

1. Re-colony PCR of transformed vector and full length colicin.

• Run gel.

8/08/16

1. Re-PCR amplify full length colicin E3 with forward RBS primer and reverse HA tag.

2. Digest XbaI + Pst.

• Run gel.

9/08/16

1. Re-digest whole colicin E3 and E3 immunity-Asr construct.

2. Re-ligate full colicin E3 and E3 immunity-Asr overnight.

10/08/16

1. Transform ligation of full E3 and immunity-promoter contruct.

11/08/16

1. No grown on any transformations.

12/08/16

1. Transform Full E3 into JM110.

2. Use this as a template for PCR.

13/08/16

1. Re-digest Pbad and colicin Ia-ssp1, colicin Ia Sa584 constructs.

2. Re-ligate these constructs.

15/08/16

1. Make competent cells.

2. Glycerol stock bricks in -80 frezzer.

16/08/16

1. Ligation of Asr and AcrRA colicins iA immunity with full length Colicin iA

1. Included vector only, 2:1 and 3:1

2. Transformed into JM110

22/08/16

1. Weighing and inoculation of chicken feed.

23/08/16

1. Storage of chicken feed.

• One set in fridge and one set under nitrogen.

24/08/16

1. Chicken feed killing experiments.

25/08/16

1. First longevity experiment for fridge stored feed.

26/08/16

1. Production and testing of DH5α and JM110 competent cells.

· Tested on chloramphenicol, ampicillin and plain agar.

1/09/16

1. Second longevity experiment for fridge stored feed.

2. First longevity experiment for nitrogen stored feed.

8/09/16

1. Third longevity experiment for fridge stored feed.

2. Second longevity experiment for nitrogen stored feed.

15/09/16

1. Fourth longevity experiment for fridge stored feed.

2. Third longevity experiment for nitrogen stored feed.

22/09/16

1. Fifth longevity experiment for fridge stored feed.

2. Fourth longevity experiment for nitrogen stored feed.

29/09/16

1. Sixth longevity experiment for fridge stored feed.

2. Fifth longevity experiment for nitrogen stored feed.

Lab Book - Rachel

Week 1 - 21/6/16

1. Familiarisation with methods and techniques

2. Restriction digest of AcrRA Bile Salt promoter

• Digested with Spe1 and Pst1
• Run on a gel

22/6/16

1. Restriction digest of Asr and GadA pH sensitive promoters

• Digested with Spe1 and Pst1
• Total volume = 50μl
• Alkaline phosphatased
• Ran on gel then gel extracted
• Nanodrop concentrations

o GadA = 22.9ng/μl

o Asr = 21.0ng/μl

2. Restriction digest of RamA

• Digested with EcoR1 and Pst1
• Total volume = 30μl
• Ran on a gel

3. Ligation of RamA with pSB1C3 vector cut with EcoR1 and Pst1

• Ligated overnight

Week 2 - 27/6/16

1. Restriction digest RamA in pSB1C3

• Digested with Xba1 and Pst1
• Total volume = 30μl
• Nanodrop concentration = 13.1ng/μl

2. Restriction Digest GFP

• Restriction digest with Xba1 and Pst1
• Total volume = 50μl
• Nanodrop concentration: 9.7 ng/ul

3. Ligation of RamA with RBS vector

• Included vector only control, 2:1 and 3:1 reaction
• Ligated overnight

28/6/16

1. Restriction digest of Colicin E3 immunity

• 2 reactions

o Digested with EcoR1 and Pst1

o Digested with Xba1 and Pst1

• Nanodrop concentrations

o EP digest = 12.7ng/μl

o XP digest = 5.2ng/μl

2. Ligation of pSB1C3 RBS with E3 immunity (cut XP)

• Included vector only control, 2:1 and 3:1 reactions
• ligated for 3 hours
• Transformed into JM110

29/6/16

1. Miniprep of pSB1C3

• Nanodrop concentration

o 72.2ng/μl

o 102.1ng/μl

2. Restriction digest of pSB1C3

• Digested with EcoR1 and Pst1
• Total volume = 50μl
• Alkaline phosphatased
• Run on gel

30/6/16

1. Miniprep E3/RBS overnight

2. PCR of Colicin E9 and iA immunity gblocks with primers

• Dry gblocks resuspended in 50μl of water
• Primers made up with water the diluted 1/10
• Ran on gel

1/7/16

1. Restriction digest of Colicin iA and E9 immunities

• Digested with EcoR1 and Pst1
• Total volume = 30μl

Week 3 - 4/7/16

1. Restriction digest RBS-GFP

• Xbal-Pst
• Nano drop GFP = 9.7 ng/ul

2. Colony PCR acrRA

• Run on gel

5/7/16

1. Ligations with GFP

• acrRA- GFP ligation
• gadA-GFP ligation
• asr-GFP ligation

2. Transformations into JM110 cells

7/7/16

1. Colony PCR of ligations from the 05/07

2. Over night cultures of promoter-GFP constructs from transformations

8/7/16

1. Miniprepped the over nights:

• Nanodrop concentrations:

o gadA-GFP colony 1: 115.2 ng/ul

o gadA-GFP colony 2: 256.3 ng/ul

o gadA-GFP colony 3: 226.2 ng/ul

o gadA-GFP colony 4: 172.5 ng/ul

o asr-GFP colony 1: 170.4 ng/ul

o asr-GFP colony 2: 134.4 ng/ul

o asr-GFP colony 3: 149.7 ng/ul

o asr-GFP colony 4: 127.9 ng/ul

2. Samples prepared for sequencing with forward and reverse primers

3. Restriction digest for promoters asr and gadA

• Cut with Xbal-Pst
• Alkaline phosphetased

9/7/16:

1. Transformation of acrRA from kit plate

• Suspend part from kit plate in 50ul H₂O (sterile) and keep as stock
• Transform into JM110 cells

2. Run gel of restriction digests of asr and gadA

3. Over nights of asr and gadA

10/7/16

1. Miniprep over nights of asr and gadA

2. Over night cultures of acrRA transformations

Week 5 - 11/7/16

1. Miniprep acrRA over night cultures

2. Restriction digest acrRA with Xbal-Pst

3. Run digest on gel.

4. Ligation of acrRA with GFP

5. Transformation of ligation into JM110 cells

6. Over night of gadA-GFP and asr-GFP and acrRA-GFP

12/7/16

1. Mini-prep over nights:

• Asr-GFP
• gadA-GFP
• acrRA-GFP

2. Send samples from above table for sequencing

3.

4. Transformations:

· gadA-GFP à MG1655 cells

· asr-GFP à MG1655 cells

· acrRA-GFP à MG1655 cells

14/7/18

1. Preparation of MOPS buffered LB

• 900ml 10x MOPS to 10ml LB
• Adjust pH with concentrated HCl or NaOH

· Filter sterilise - ready for use.

2. Over night cultures from JM110 plates ready for glycerol stocking

-------------------

15/7/16

1. Restriction digest of RBS pSB1C3

• Digested with Spe1 and Pst1
• Total volume = 50μl

Week 6 - 18/7/16

1. PCR of SA584 immunity

2. Restriction digest of Colicin iA and E9 MCS

• Digested with BamH1 and Nhe1
• Ran on gel

3. Ligated Colicin iA and E9 MCS with toxins Ssp1, Ssp2 and PPFP

• Included vector only, 2:1 and 4:1 reactions
• Transformed into JM110

19/7/16

1. Restriction digest of pUniprom

• Digested with BamH1 and Sal1
• Total volume = 50μl

2. Ligation of pUniprom with PPFP immunity

• Included vector only, 4:2 and 7:2 reactions

3. PCR of full colicins and immunities with RBS and Ha tag primers

• Ran on gel

21/7/16

1. Restriction digest of Colicin iA and E9 with toxic warheads

• Digested with Pst1
• Ran on gel to check size of band
• Sent for sequencing:

o ColiA Ssp1 B/C

o ColiA Ssp2 B/C

o ColE9 Ssp1 A/C

o ColE9 Ssp2 B/C

o ColE9 PPFP B/C

Week 7 - 25/7/16

1. PCR of Col MCS with toxins with RBS primer

2. PCR of PPFP immunity

3. Quick change of Col E9 PPFP immunity

27/7/14

1. PCR of Colicin MCS with toxins ith RBS

• Ran on gel

2. Colony PCR of ColiA PPFP toxin

• 2:1 – samples 1,3,6 sent for sequencing
• 3:1 – samples 1,4,6 sent for sequencing

30/7/16

1. Restriction digest of PPFP immunity

• Digested with BamH1 and Sal1
• Total volume = 30μl

2. Ligation PPFP immunity with pUniprom

• Included vector only, 2:1 and 4:1 reactions

Week 8 - 1/8/16

1. Transformation of Ssp1/2 immunities

2. Made competent cells with Ssp1/2 immunities

2/8/16

1. Ligation of PPFP immunity with pUniprom

• Included vector only, 2:1 and 3:1
• Transformed into JM110

3/8/16

1. Colony PCR of transformation of PPFP immunity with pUniprom

• Took overnights

2. Ligation of RBS colicins with toxins with Asr promtoter

• Included vector only, 2:1 and 4:1
• Transformed into JM110
• Did not work

4/8/16

1. Overnights of PPFP immunity colony PCR did not grow

2. Quick change of Colicin iA immunity in Asr and AcrRA promoter

3. Tested Ssp1/2 immune competent cells

4. Overnights

• ColiA Ssp1 pSB
• ColiA Ssp2 pSB
• ColE9 Ssp1 pSB
• ColE9 Ssp2 pSB
• Col iA MCS pSB
• ColE9 MCS pSB
• Col E9 PPFP quick change x3
• PPFP immunity x3
• ColiA SA584 in MG for quick change

5/8/16

1. Overnight that did not work

• ColE9 PPFP
• PPFP immunities

Week 8 - 8/8/16

1. Quick change of ColiA PPFP

2. Restriction digest of pBAD and Colicins with toxins

• pBAD digested with EcoR1 and Xba1
• Colicin toxins digested with EcoR1 and Spe1
• Total volume = 30μl

3. PCR up colE9 and iA PPFP with RBS

• Ran on gel
• Col E9 did not work

4. Ligation of pBAD and Colicin toxins

• Included vector only, 2:1 and 3:1 reactions
• Ligated overnight

9/8/16

1. Transformed pBAD Colicin iA and E9 Ssp1/2 ligations into MG1655

2. PCR up Colicin E9 and iA PPFP with RBS

3. Restriction digest of ColiA PPFP

• Digested with EcoR1 and Spe1
• Total volume = 40μl

4. Ligation of coliA PPFP with pBAD

• Included vector only, 2:1 and 3:1 reactions
• Transformed into MG1655

10/8/16

1. No grown on any transformations

11/8/16

1. Ligation of Colicin iA and E9 Ssp1/2 and IA PPFP(not quick changed) with pBAD

• Included vector only, 2:1 and 3:1 reactions
• Transformed in MG1655

2. Made MG1655 and JM110 competent cells

3. Overnights for western blot

• pSB Asr
• pSB Asr E3 immunity
• pSB Asr iA immunity
• pSB and pUni
• pSB AcrRA and pUni RamA
• pSB AcrRA ColiA im and pUni
• pSB AcrRA ColiA im and pUni RamA
• pSB AcrRA ColE3 im and pUni
• pSB AcrRA ColE3 im and pUni RamA

12/8/16

1. Colicin toxins in pBAD grew, no vector only growth

• Overnights taken

2. Western blot of Asr and AcrRa coliA and E3 immunities

• OD’s of overnights

o pSB Asr – 3.43A

o pSB Asr E3 immunity – 3.72A

o pSB Asr iA immunity – 3.79A

o pSB and pUni – 3.45A

o pSB AcrRA and pUni RamA – 4.41A

o pSB AcrRA ColiA im and pUni – 3.36A

o pSB AcrRA ColiA im and pUni RamA – 3.28A

o pSB AcrRA ColE3 im and pUni – 3.26A

o pSB AcrRA ColE3 im and pUni RamA – 4.81A

13/8/16

1. Colicin toxins in pBAD overnights were miniprepped and sent for sequencing

Week 9 - 15/8/16

1. PCR of

• Col E9 Ssp2 2:1
• Col iA PPFP 3:1
• Col E9 Ssp1 3:1
• Col iA Ssp1 2:1
• Col iA Ssp1 3:1

16/8/16

1. Ligation of Asr and AcrRA colicins iA immunity with full length Colicin iA

• Included vector only, 2:1 and 3:1
• Transformed into JM110

17/8/16

1. Overnights of 16/8/16 transformations

18/8/16

1. Miniprepped overnights and sent them for sequencing

21/8/16

1. Transformations into MG1655 for western blot

• ColiA Ssp2 3:1
• ColE9 Ssp2 3:1
• ColE9 Ssp2 2:1
• ColE9 Ssp1 2:1
• ColiA Ssp2 2:1
• pBAD (for negative control)
• ColiA MCS (for negative control)
• Plated onto 0.5% glucose kanamycin plates

Week 10 - 22/8/16

1. Overnights of transformations

• Added 0.5% glucose to overnight

23/8/16

1. Western blot of Colicin toxins in pBAD

• OD’s of overnights

o ColiA Ssp2 3:1 – 1.625A

o ColE9 Ssp2 3:1 – 1.536A

o ColiA Ssp2 2:1 – 1.586A

o ColiA MCS 3:1 – 1.591A

o ColE9 Ssp1 2:1 – 1.528A

o ColE9 Ssp2 2:1 – 1.595A

24/8/16

1. Overnights for western blot

• ColiA MCS
• ColiA Ssp2 2:1
• ColiA Ssp2 3:1

25/8/16

1. Subculture and induction of coliA MCS and Ssp2 for western blot

• OD’s of overnights

o ColiA MCS – 0.67A

o ColiA Ssp2 2:1 – 0.63A

o ColiA Ssp2 3:1 – 0.61A

26/8/16

1. Western blot of pre-induced and induced ColiA and Ssp2 samples

30/8/16

1. Made four 10% acrylamide gels

2. Overnights for western blot

• pSB Asr E3 immunity
• pSB Asr iA immunity
• pSB Asr (Negative control)
• pBAD ColE9 Ssp1
• pBAD ColE9 Ssp2

3. Overnights for sequencing

• ColiA Ssp1 x 6

31/8/16

1. Western blot of Asr iA and E3 immunities

• OD of overnight

o Asr – 3.61A

o Asr iA immunity – 3.47A

o Asr E3 immunity – 3.85A

2. Western blot of ColiA Ssp2 supernatant

· Supernatant was TCA precipitated and concentrated

3. Subculture and induction of ColE9 Ssp1 and Ssp2

• OD of overnights

o ColE9 Ssp1 – 0.18A

o ColE9 Ssp2 0.17A

1/9/16

1. Western blot of ColiA Ssp2 supernatant and ColE9 Ssp1 and 2 pellets

2/9/16

1. Prepare samples of Asr and AcrRA coliA immunity for western blot

• Overnight OD’s

o Asr ColiA immunity – 4.00A

o AcrRA ColiA immunity – 4.17A

2. Plate toxicity to test toxicity of colicins with toxins

• OD’s

o ColiA MCS – 1.60A

o ColiA Ssp2 – 4.29A

o ColE9 Ssp1 – 1.98A

o ColE9 Ssp2 – 2.81A

· Plated onto 0.2% glucose, 0.2% arabinose, 0.4% arabinose

6/9/16

1. Transformed pBAD into MG1655

2. Transformed full length coliA pSB into JM110

3. Overnights of pSB Asr iA and E3 immunities and pSB Asr

7/9/16

1. Subculture of Asr E3/iA immunities at pH5 and pH7

• OD’s of overnights

o pSB Asr – 3.46A

o pSB Asr iA immunity – 3.77A

o pSB Asr E3 immunity – 3.88A

• Cultured overnight

2. Western block of ColE9 Ssp1 and Ssp2 purified supernatant

• Blot was blank

3. Overnights ColiA MCS and ColiA Ssp2

8/9/16

1. Subculture of ColiA MCS and ColiA Ssp2

• OD’s of overnights

o ColiA MCS – 4.29A

o ColiA Ssp2 – 2.40A

· Added 250μl of Ssp2 and 125μl of MCS so that they were roughly the same OD and could be induced at the same time

• Cultures all day

2. Asr ColE3 and iA immunity subculture

• OD’s after overnight subculture

o pH7

§ Asr – 4.2A

§ ColiA immunity – 4.0A

§ ColE3 immunity – 4.5A

o pH5

§ Asr – 0.4A

§ ColiA immunity – 0.05A

§ ColE3 immunity – 0.4A

• Prepared samples for blotting

9/9/16

1. Western blot of Asr E3/iA immunities at pH5 and pH7

15/9/16

1. Overnights

• pBAD MG1655
• pBAD ColE9 Ssp1 MG1655
• pBAD ColE9 Ssp2 MG1655
• MG1655

2. Lysis of ColiA Ssp2 to test toxicity

• Cells were scintillated how 1.5 minutes
• On plate

o 1 – ColiA MCS Glucose

o 2 – ColiA MSC Arabinose

o 3 – ColiA Ssp2 Glucose

o 4 – ColiA Ssp2 Arabinose

o 5 – Ampicilin

o 6 – PBS

• 20μl of each was plated
• Incubated overnight

16/9/16

1. Subculture of colE9 Ssp1 and Ssp2

• Cultured for 8 hours
• Samples prepared for blotting

2. Lysis plates did not show toxicity

20/9/16

1. Lysis of ColiA Ssp2 to test toxicity

• Cells were scintillated how 1.5 minutes
• On plate

o 1 – ColiA MCS Glucose

o 2 – ColiA MSC Arabinose

o 3 – ColiA Ssp2 Glucose

o 4 – ColiA Ssp2 Arabinose

o 5 – Ampicilin

o 6 – PBS

• 20μl of each was plated
• Incubated overnight

2. Lysis of ColE9 Ssp1 and Ssp2 to test toxicity

• Cells were scintillated how 1.5 minutes
• On plate

o 1 – pBAD Arabinose

o 2 – ColE9 Ssp1 Arabinose

o 3 – ColE9 Ssp2 Arabinose

o 4 – Ampicilin

o 5 - PBS

• 20μl of each was plated
• Incubated overnight