Experiments and Protocols

Our Work Flow

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Colony PCR Protocol

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1. Add the following reagents in an Eppendorf (keep all substances on ice whilst preparing the mixture).

Reagent	Amount for 10 reactions (µl)
PCRBIO Taq 2x polymerase red mix	125
Forward primer 10 µM	10
Reverse primer 10 µM	10
Sterile Distilled Water	105
Total Volume	250

2. Scratch some colony from a plate, and swirl the pipette tip in the mixture a few times before disposing of it.
3. In thermocycler:

Step	Cycles	Temperature (°C)	Time (s)
Initial Denaturation	1	95	300
Denaturation	35	94	30
Annealing	35	59.5-64.5 (depending on the primer's Tm)	45
Extension	35	72 *	20 per kilobase
Final Extension	1	72	300
Hold	1	4	∞

*Varies depending on the length of the insert

Miniprep

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Followed protocol from FastGene plasmid minikit / Omega E.Z.N.A plasmid minikit

PCR (Including Clean Up)

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1. Produce 50 µl of PCR mixture using the following volumes:

Reagent	Volume (µl)
Q5 2X Hot Start Master Mix	12.5
Forward primer	2.5
Reverse primer	2.5
Template DNA (conc. 100pg/µl)	1.0
Sterile Distilled Water	31.5
Total Volume	50

(Keep all reagents on ice while producing the mixture)

2. Place the mixture into a thermocycler with the lid preheated to 105 °C. Follow the program detailed below:

Step	Cycles	Temperature (°C)	Time (s)
Initial Denaturation	1	95	300
Denaturation	25-35	94	30
Annealing	25-35	59.5-64.5 (depending on the primer's Tm)	45
Extension	25-35	72	105
Final Extension	1	72	300
Hold	1	4	∞

3. Followed protocol from Monarch PCR & DNA Cleanup Kit for Cleanup

Transformation Protocol

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credited to New England Biolabs¹

1. Thaw competent cells on ice.
2. Chill approximately 5 µl of plasmid DNA in a 1.5 ml microcentrifuge tube.
3. Add 50 µl of competent cells to the DNA. Mix gently by pipetting up and down or flicking the tube 4-5 times to mix the cells and DNA. Do not vortex.
4. Place the mixture on ice for 30 minutes.
5. Heat shock at 42°C for 30 seconds.
6. Add 950µl of room temperature LB media to the tube.
7. Place the tube at 37°C for 60 minutes, shake at 250rpm.
8. Spread 50-100 µl of the cells and ligation mixture onto the agar LB plates containing antibiotics for selection.
9. Incubate overnight at 37°C.

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¹www.neb.com. (n.d.). Transformation Protocol | NEB. [online] Available at: https://www.neb.com/en-gb/protocols/2012/05/21/transformation-protocol.

Golden Gate Assembly

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Used this protocol: https://www.neb.com/en-gb/protocols/2018/10/02/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-mix-e1601

Protein Purification

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Step Up

BL21 LEMO was used as the protein expression strain.

1. A colony was scraped from a plate, inoculating a 10 ml LB (ampicillin) overnight.
2. The following morning, this was used to inoculate a 500ml LB (ampicillin) culture.
3. These were then shaken at 37°C and ~180rpm, with regular OD measurements until the OD was between 0.4 and 0.6.
4. IPTG was added at a final concentration 500µM, and then it returned to shaking at 180 rpm at 16°C overnight.

Lyse the cells

1. Spin induced culture down at 4500 rpm for 15 minutes in falcon tubes of 50ml until supernatant is clear.
2. Discard the supernatant and resuspend all pellets in a single 30ml of binding buffer.
3. Sonicate this on ice for a total sonification time of 6 minutes in 30 second intervals with a break for 30 seconds in between intervals.
4. Centrifuge the cell lysate at 21500rpm for 15 minutes at 5C using Rotor-JA 25.50

Prepare the Nickel Column

All the following steps use the method of allowing the liquid to flow through. To speed this up, the column was inserted into a falcon tube with a hole in the lid and this was centrifuged at 450 rpm for 30 seconds to increase the speed of flow-through.

1. Into an empty P10 column fill with 5ml of Sepharose Bead slurry (for the rest of the protocol 5ml will be referred to as CV(Column volume))
2. When the column is not in use keep the lid and tip of the P10 column on and store the bead slurry in 100% ethanol
3. When the column is required it needs to be washed and charged prior to use:
   1. Wash the column through with 4CVs of MQ H2O
   2. Charge the column by adding 4CVs of NiSO4
   3. Wash the column through with 4CVs of MQ H2O
   4. Equilibrate the column by washing through with 4CVs of binding buffer
4. Place the cap and lid of the column back on when not in use

Load the lysate

1. Gently filter sterilise the supernatant of the spun lysate onto the top of the column slurry.
2. Allow this to completely flow through the column and collect this in a falcon tube labelled FT.

Binding and Washing

1. Load 4CVs of binding buffer into the column and allow to flow through, collecting it in a falcon tube labelled BB
2. Load 4CVs of wash buffer into the column and allow to flow through, collecting it in a falcon tube labelled WB

Elution

1. Load 1ml of elution buffer to fully flow through and collect in a 1.5ml eppendorf labelled E1
2. Repeat this until you have 10x1ml elutions (e.g. labelled E1, E2, E3….)
   1. By eluting slowly and running an SDS-PAGE you can work out which fractions have the protein to pool these

Washing and storage of the Nickel column

1. Wash the column in 4CVs of EDTA to scrub the Nickel off the beads
2. Wash the column through with 4CVs of MQ H2O
3. Store in 100% ethanol until next use

SDS-PAGE: Protein Electrophoresis

1. Take 10µl of FT, BB, WB and E1-10 from the Nickel Column Purification and take 10µl from each and place into a fresh Eppendorf
2. Add 10µl of 2x SDS Sample Buffer
3. Carefully load your samples into the wells of the SDS gel and load 5µl of ladder into the required well
4. Run the gel at 150V for 1 hour
5. Carefully extract the gel from the plastic casing and place the gel in a Tupperware container and cover in QuickBlue stain
6. Place on a slow rolling shaker for at least 10-15 minutes but for best results leave in the stain longer
7. Use a lightblock to visualise the gel

Desaltification

1. Into a PD-10 Column fill with 8ml of Sephadex G-25 resin
2. Fill up the column with 25ml (will need to be done in several steps) of equilibration buffer and allow to enter the packed bed completely
3. Discard the flow-through
4. Add 2.5ml of sample to the column
5. Discard the flow through
6. Elute with 3.5ml of buffer and collect the elute.
7. Repeat steps 4 to 6 until all the sample have been passed through the column
8. Samples were then spun in a Sample Concentration Column to have a final volume of 1-1.5ml

Size Exclusion

1. Connect a Superdex 200 Increase 10/300 GL column to an ÄKTA chromatography system
2. Equilibrate the column by running 2 column volumes of protein storage buffer (25 mM HEPES, 100 mM NaCl, pH 7.4) through the column
3. Samples were concentrated down to 500 µl of volume in a centrifugal spin concentrator with a 30,000 MW cutoff
4. Samples were then injected onto the column with a 0.75 ml/min flow rate, and fractions were collected every 1 ml after injection
5. Fractions corresponding to a peak in UV absorbance were run on an SDS gel to check which peak contained our purified protein

Competent Cells

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Through the use DH5α cells, we expect to get a high competency with these (at least 1 x 10⁶ per µg DNA). Does not require antibiotics (so be careful to work aseptically)

1. Put up a 5ml overnight of DH5α in LB, at 37°C shaking.
2. In the morning; inoculate a flask of 400ml LB with the whole 5ml overnight, and shake at 37°C until the OD550 = 0.48. At this point, place on ice and process when completely chilled - can be later in the day.
3. Spin down the whole contents of the flask in 50ml falcon tubes for 5mins at top speed (4500rpm).
4. Combine the pellets in a total of 2 x 50ml ice cold TFB1 (i.e. 4 pellets per 50ml TFB1). Ensure completely resuspended.
5. Place on ice for 10mins.
6. Spin down for 5mins at 4500rpm, discard supernatant, and resuspended each pellet in 10ml ice cold TFB2 (i.e. now have 20ml total).
7. Place on ice for 15mins
8. Put 200µl aliquots into sterile coloured tubes (any colour; no need to label), and store immediately at -80°C.

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How to make TFB1 and TFB2

TFB1:

Reagent	Volume (ml) for 125ml
300mM KAc (autoclaved)	12.5
1M RbCl (filter sterilised)	12.5
100mM CaCl2 (autoclaved)	12.5
500mM CaCl2 (autoclaved)	12.5
glycerol (autoclaved)	18.5

pH to 5.8 with acetic acid then make up to 125ml with sterile H₂O and filter sterilise into a sterile bottle. Store at 4°C.

TFB2:

Reagent	Volume (ml) for 50ml
100mM MOPS (autoclaved)	5.0
750mM CaCl2 (autoclaved)	5.0
100mM RbCl (filter sterilised)	5.0
glycerol (autoclaved)	7.5

pH to 6.5 with KOH then make up to 50ml with sterile H₂O and filter sterilise into a sterile bottle. Store at 4°C.

PNPP Protocol

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For us to characterise the activity of PafA and the variants we created, we decided to utilise PNPP. Phosphatase enzymes can hydrolyse PNPP into inorganic phosphate and pNP, the latter of which is yellow. This crucial property allows us to measure the absorbance at 405nm on a spectrophotometer or plate reader and convert these absorbance values into the concentration of pNP. This enables us to gather information such as V_max and K_m to determine the effect of different conditions on enzyme activity.

Before we conducted the assays we needed to make up reaction buffer for the enzyme and substrate buffer for PNPP which consist of:

Substrate buffer
100mM Tris HCl pH7.4
200mM NaCl

Reaction Buffer
100mM Tris
200mM NaCl
100µM ZnSO4

The starting assay for all of the characterisations was a kinetics assay, which consisted of varying concentrations of PNPP to enable us to create a Michaelis Menten graph to determine the kinetic properties of the enzyme. Figure 1 depicts the process which we went through for each of the kinetics assays. For the variants, we typically used 11.1nM or 111.1 nM of enzymes in the rows.

Figure 1: Stages of Kinetic Assay. Using a 96-well plate, create the appropriate array of chemicals as shown, and using a multichannel pipette, add the contents from Row A to all other rows after closing the lid, and add it to a plate reader, measuring at 30 seconds for 90 minutes.

For pH assays, we used a pH meter to make up PNPP to a variety of different pH’s (sodium acetate (pH 4, 5, 6 and 6.5); Tris/HCl (pH 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10)) and pipetted this into row A instead of having a serial dilution of PNPP.

For the temperature assays, we used a hot block at temperatures ranging from 15 to 90°C using the protocol in Figure 2 The stop solution was made up of 1.1M NaOH, 25mM EDTA, 500mM K₂PO₄.

Figure 2: The stages of pH Assay. This goes through the stages of preheating the constituents, adding the appropriate solution to the enzyme and then measuring the absorbance after the stop solution was added.

For the inhibition assays, we utilised Pi ranging from 5mM to 0.00244mM. We added Pi to the assay and then incorporated PNPP on the top row, utilising a serial dilution. This way each column had a decreasing concentration of Pi to measure the level at which product inhibition occurs. We did 2 plates, the first plate being PafA and PhoA inhibition levels being measured.

The second plate contained the variants; 30.1, 50.2 and 70.2. Each row from B to G contained 20ul of enzyme across the entire plate, A contained the Pi-PNPP-RB mixture and H contained simply just RB to act as a control.

Redesign Protocol

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Use AI guide linked here

Media Protocols

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LB Broth:

• Brand used → FORMEDIUM
• 25 g is suspended in 1 L of distilled water, then the solution is autoclaved

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LB Agar:

• Brand used → FORMEDIUM
• 40 g is suspended in 1 L of distilled water, then the solution is autoclaved

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Agarose Gel:

• Agarose brand used → AMRESCO
• (50X) TAE brand used → Thermo Fisher Scientific
• 1% agarose gels are usually made (unless stated otherwise) - 1 g of agrose dissolved in 100 ml of 1X TAE

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Induction:

• IPTG brand used → Thermo Fisher Scientific
• 500 µM of IPTG (dioxane-free) is used

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TAE Solution:

• (50X) TAE brand used → Thermo Fisher Scientific
• The Solution is diluted to a 1X solution by adding 20 ml TAE to 980 ml distilled water