Editing S30 E. coli extract (section)

== <span style="font-family: arial;">Protocol 4</span> ==

[http://ajournal.co.uk/pdfs/volume1/Vol.1%20(1)%20Article%205.pdf http://ajournal.co.uk/pdfs/volume1/Vol.1%20(1)%20Article%205.pdf]

Cell-free protein synthesis for analysis by NMR spectroscopy,&nbsp;British Journal of Science 45 September 2011, Vol. 1 (1)

'''3.1.1. Day One: Buffer preparation and overnight culture&nbsp;'''

1. Prepare and autoclave stock solutions of glucose (2 M, 224 mL), thiamine (1 mg/mL, 200 mL), 10X S30 buffer (2 L) and 50% PEG 8000 (2 L) as listed in the materials section. Stir the mixture of 50% PEG 8000 at room temperature overnight. Autoclave the stock solutions of glucose, thiamine and 10X S30 buffer, 1 L of LBT medium and 20 L of MQ water for diluting the 10X S30 buffer stock. Autoclave a large glass funnel and two 250 mL measuring cylinders required for measuring the glucose and thiamine solutions to be added to the medium on day three.

&nbsp;LBT: 10 g/L tryptone, 5 g/L yeast extract, 5 g/L sodium chloride, 2.5 mL/L 1 M sodium hydroxide, 0.5 mg/L thymine

2. Grow a 10 mL culture of '''BL21 DE3 star''' cells (invitrogen) in LBT overnight at 37 oC. If the BL21 DE3 Rosetta pRARE strain is used, include 33 ug/mL of chloramphenicol.

3. After autoclaving, store MQ water and 10X S30 buffer at 4 oC.

'''3.1.2. Day Two: Z-medium preparation and overnight 1 L culture'''

1. Prepare 5 L of 4X Z-medium buffer.

2. Add 5 L of Z-medium concentrate, 4.5 mL of antifoam and 14 L of MQ water into the fermenter. This gives a total volume of 19 L. When 1 L of overnight culture is added to this the following day, 20 L of 1X Zmedium will result.

4X Z-medium: 165 mM potassium dihydrogen phosphate, 664 mM potassium phosphate dibasic, 40 g/L yeast extract

3. Sterilise the medium at 121 oC using the sterilisation function of the fermenter.

4. Calibrate the pO2 electrode of the fermenter to 0% after 25 min at 121 oC.

'''3.1.3. Day Three: 20 L culture and processing of cell pellets'''

1. Equilibrate the fermenter at 37 oC, with 100% air and mixing rate at 395 rpm.

2. Prior to addition of the overnight culture, remove a sample from the outlet at the base as a blank for later measurement of the absorbance of the culture at 595 nm (OD).

3. Add the 1 L overnight culture, 224 mL of 2 M glucose and 200 mL of 1 mg/mL thiamine to the Z-medium with sterile glass funnel. .

4. Allow these to mix before removing a time zero sample for OD measurement.

5. Remove samples for OD measurements every 30 minutes until the culture reaches an OD of 3. This density should be achieved after 3-4 hours.

6. Fill a large sink or tub with a large amount of crushed ice in preparation for the next steps.

7. Drain the culture into 5 L conical flasks and immediately place them on ice. Weigh empty 1 L tubes.

8. Harvest the cells by centrifugation at 4 oC for 12 min at 10,000 g in 1 L tubes. Use pre-weighed tubes so that the pellet mass can be determined later.

9. Resuspend the cell pellets in 400 mL of 1X S30 buffer A. Centrifuge the resuspended cells for 10 min at 10,000 g and 4 oC.

10. Remove the supernatant and pack the pellet by centrifugation for a further 5 minutes. Remove the remaining supernatant with a pipette.

11. Snap freeze the pellets in liquid nitrogen, then store them at –80 oC

'''3.1.4. Day Four: Preparation of the S30 extract''' 1. Remove the pellets from the freezer and defrost slowly on ice for at least one hour prior to resuspension.

2. Resuspend the pellets in 400 mL of S30 buffer A, and centrifuge for 12 min at 10,000 g.



4. Resuspend the pellets in 1.3 mL of S30 buffer A&nbsp;per gram of cells.

5. Subject the suspension to a single pass in a French press at a pressure of 6,000 psi. If the lysate is very viscous pass it through the French press again.

6. Centrifuge the lysed cells at 5,200 g at 4 oC for 15 min to remove any cell debris in 1 L tubes. Transfer the supernatant to clean tubes.

7. Centrifuge the supernatant at 30,000 g at 4 oC for 1 h to produce the S30 fraction.

8. Prepare 3 batches of 3 L each of S30 buffer B. Dialyse the S30 fraction in Spectrapor # 4 dialysis tubing against each 3 L batch of S30 buffer B for one hour at 4 oC, i.e. for three hours in total.

S30 buffer B: 1X S30 buffer containing 1 mM DTT

8a. Optional: Take 1/3 of S30 extract before the following concentration step. Snap-freeze and store it at -80 oC.

9. Transfer the dialysis tubes containing the S30 fraction to a large evaporating dish and dialyse against two batches of 1L of 50% PEG 8000. Place the dish on a shaking tray to speed dialysis, and leave for 2 hours at 4 oC.

10. Change the 50% PEG buffer and repeat until the volume of the extract has been reduced to approximately half volume of the S30 extract (about 2h). It is measured by visual comparison with dialysis tubing containing 60 mL of water.

11. The concentrated S30 fraction is then dialysed against a further 3 L of 1X S30 buffer B for 15 minutes to remove the residual PEG 8000 buffer.

12. Measure the volume of the extract. Centrifuge the S30 extract if the level of precipitation is high.

13. Dispense the extract into 1 mL aliquots in 1.5 mL Eppendorf tubes.

14. Snap-freeze the aliquots in liquid nitrogen and store at -80 oC.

12a. Place the dialysis bag containing the S30 extract into a further 2 L of S30 buffer C for dialysis overnight at 4 oC.

13a. Place the dialysis bag containing the S30 extract into a 250 mL Pyrex glass bottle, filled with the S30 buffer C from the overnight dialysis. Prewarm that S30 buffer C to 42 oC.

For each new batch of S30 extract, the concentrations of magnesium acetate and the extract itself must be optimized. A series of reactions using between 9-25 L S30 extract per 100 L reaction mixture and between 15-25 mM magnesium acetate should be sufficient to find the optimal concentrations of each.

{|
|-
| Reagent&nbsp;
| Stock conc.
| MW
| Dry weight (g)<br/>
| Stock volume (mL)<br/>
| Final conc. in reaction mixture<br/>
| Supplier<br/>
| Storage (oC)<br/>
|-
| HEPES
| 2.0 M pH 7.5
| 238.3
| 95.35
| 200
| 55 mM
| Sigma
| RT
|-
| DTT
| 0.5 M
| 154.25
| 3.85
| 50
| 1.7 mM
| Sigma
| –20
|-
| ATP
| 96 mM
| 551.14
| 0.529
| 10
| 1.2 mM
| Sigma
| –80
|-
| rNTP
| 25 mM each
| 527.1
| 0.132
| &nbsp;
| 0.8 mM
| Sigma
| &nbsp;
|-
| &nbsp;
| &nbsp;
| 523.18
| 0.131
| &nbsp;
| 0.8 mM
| Sigma
| &nbsp;
|-
| &nbsp;
| &nbsp;
| 484.14
| 0.121
| &nbsp;
| 0.8 mM
| Sigma
| &nbsp;
|-
| Total
| &nbsp;
| &nbsp;
| &nbsp;
| 10
| 0.8 mM
| &nbsp;
| –80
|-
| Cyclic AMP
| 100 mM
| 351.2
| 0.351
| 10
| 0.64 mM
| Sigma
| –80
|-
| Folinic Acid&nbsp;
| 10 mM
| 511
| 0.0511
| 10
| 68 µM
| Sigma
| –80
|-
| Ammonium acetate (monohydrate)
| 9.2M
| 77.08
| 35.5
| 50
| 27.5 mM
| Ajax Finechem
| –80
|-
| tRNA
| 17.5 mg/mL
| &nbsp;
| 0.0175
| 1
| 0.175 mg/mL
| Roche
| –20
|-
| Creatine kinase
| 10 mg/mL
| 18 kDa
| 0.1
| 10
| 250 µg/mL
| Roche
| –80
|-
| colspan="2" | T7 RNA polymerase
| &nbsp;
| &nbsp;
| &nbsp;
| 93 µg/mL
| &nbsp;
| –20
|-
| pKO1166
| &nbsp;
| &nbsp;
| &nbsp;
| &nbsp;
| 32 µg/mL
| &nbsp;
| –20
|-
| Plasmid
| &nbsp;
| &nbsp;
| &nbsp;
| &nbsp;
| 16 µg/mL
| &nbsp;
| –20
|-
| S30 extract
| &nbsp;
| &nbsp;
| &nbsp;
| &nbsp;
| &nbsp;~225 µL/mL
| &nbsp;
| –80
|-
| Potasisum glutamate
| 4 M&nbsp;
| 203.23
| 40.65
| 50
| 208 mM&nbsp;
| Sigma
| –80
|-
| Magnesium acetate
| 1.07 M&nbsp;
| 214.45
| 11.47
| 50
| ~19.3 M
| Ajax Finechem
| RT
|-
| Creatine phosphate
| 1 M
| 327.14
| 16.36
| 50
| 80 mM
| Sigma
| –80
|-
| L-Alanine
| 100 mM
| 89.09
| 0.089
| 10
| 2 mM
| Sigma
| –80
|}





3.3. Preparation of the amino-acid stock solutions

The amino-acid stock solutions are prepared in three groups, water-soluble, acid-soluble, and base-soluble. Each amino acid is present in the stock solutions at a 50 mM concentration. The acid-soluble and basesoluble combinations are dissolved in 1 M hydrochloric acid and 1 M potassium hydroxide, respectively, such that when they are mixed, the pH of the solutions is close to neutral. The stocks can be kept on ice but the complete mixture must be mixed at room temperature to avoid precipitation at the final near-neutral pH. For preparations with individual amino acids provided in isotope labelled form, the stocks are prepared minus the amino acid of interest. The labelled amino acid is added later to the final reaction mixture from a stock of a concentration adjusted for economical usage of the labelled amino acids (14). The concentrations of the individual 100X 15N stock solutions are also listed in Table 2.&nbsp;

{|
|-
| colspan="3" | Water soluble mixture - dissolved in 10 mL water
|-
| Amino acid
| Weight (mg)
| 15N stock (mM)
|-
| Ala
| 44.5
| 100
|-
| Arg
| 105
| 35
|-
| Gly
| 37.5
| 100
|-
| His
| 105
| 15
|-
| Lys
| 91.3
| 100
|-
| Pro
| 57.6
| n/a
|-
| Ser
| 52.6
| 100
|-
| Thr
| 59.6
| 15
|-
| Val
| 58.6
| 100
|-
| colspan="3" | Acid soluble mixture - dissolved in 10 mL 1 M HCl
|-
| Amino acid
| Weight (mg)
| 15N stock (mM)
|-
| Asn
| 66
| 35
|-
| Asp
| 66.5
| 100
|-
| Cys
| 60.1
| 35
|-
| Glu
| 73.5
| 100
|-
| Gln
| 73.1
| 100
|-
| Leu
| 65.6
| 100
|-
| Met
| 74.6
| 100
|-
| Trp
| 102
| 5
|-
| Tyr
| 90.6
| 5
|-
| colspan="3" | Base soluble mixture - dissolved in 10 mL 1 M KOH
|-
| Amino acid
| Weight (mg)
| 15N stock (mM)
|-
| Ile
| 65.5
| 15
|-
| Phe
| 82.5
| 35
|}

To same procedure is used to prepare each of the three types of amino acid stock solutions.

1. Weigh out the individual amino acids in the quantities listed in Table 2.

2. Place all of the amino acids into a 10 mL tube.

3. Add 10 mL of the appropriate solvent to the mixture and vortex until dissolved. 4. Store the amino-acid stock at -80 oC.



3.4. Carrying out the cell-free reaction

3.4.1. Preparation of the reaction chamber

1. Soak one 10 cm length of Spectrapor #2 dialysis tubing per reaction in MQ water for 5 min, then tie a knot as close as possible to one end. Trim the knot to have minimal overhang.

2. Using clean scissors, prepare one 500 L Eppendorf tubes per reaction by cutting off the lid and hinge sections. The lid is retained and the hinge is discarded. Removal of the hinge allows the tube to fit inside a 10 mL tube. The bottom 0.5 cm of the tube is then sliced off using a scalpel.

3. Insert each cut Eppendorf tube into the top of a pre-tied length of dialysis tubing, as shown in Fig. 1. The Eppendorf tube ensures the contents of the dialysis tube are readily accessible. Remove the dialysis tubing from the MQ water and insert the Eppendorf tube immediately prior to use, so that the tubing does not dry out before assembly of the complete reaction.&nbsp;

Fig. 1: Schematic drawing of the assembly of the cut Eppendorf tube in the knotted dialysis bag to provide a readily accessible container for the reaction mixture.

'''3.4.2. Preparation of the reaction mixtures'''



Table 3: 10X B solution for a 500 µL reaction with 5 mL outside buffer

{| border="0" cellspacing="0" cellpadding="0"
|-
| colspan="2" | Stock solution µL
|-
| rNTP
| 179.5
|-
| HEPES
| 162.5
|-
| ATP
| 70
|-
| Water
| 38.5
|-
| Folinic acid
| 38
|-
| Cyclic AMP
| 35
|-
| DTT
| 19
|-
| Ammonium acetate
| 17
|-
| Total
| 559.5
|}



{| border="0" cellspacing="0" cellpadding="0"
|-
| colspan="3" | Table 4: Components of a 500 µL reaction mixture and 5 mL outside buffer
|-
| colspan="3" | Stock solution Reaction mixture (µL) Outside
|-
| buffer<br/>
| (µL)
| 
|-
| 10X B
| 50
| 500
|-
| Creatine phosphate
| 40
| 400
|-
| Potassium glutamate
| 26
| 260
|-
| Magnesium acetate
| 7
| 90
|-
| L-Alanine
| 5
| 50
|-
| Amino acid mixture
| 35
| 335
|-
| Creatine kinase
| 12.5
| 500
|-
| T7 polymerase vector
| 8
| 500
|-
| S30 extract
| 112.5
| 500
|-
| Vector of interest
| 8
| 500
|-
| tRNA
| 5
| 500
|-
| Water (adjust)
| 190
| 3365
|-
| total
| 500
| 5000
|}

3.4.2.1. Mixing the amino-acid stock solutions for use in a 500 µL reaction mixture with 5 mL outside buffer

1. Pipette 37.5 µL water into a 1.5 mL Eppendorf tube.

2. Add 112.5 µL water soluble stock and pipette up and down to mix well.

3. Add 112.5 µL acid soluble stock and pipette up and down to mix well.

4. Add 112.5 µL base soluble stock and pipette up and down to mix well.&nbsp;

3.4.2.2. Preparation of the 10X B mixture

Working on ice, add the stock solutions (described in Table 1) in the quantities listed in Table 3 in a 1.5 mL Eppendorf tube.

3.4.2.3. Preparation of the reaction mixture and outside buffer

Working on ice, mix the components listed in Table 4 for the reaction mixture in a 1.5 mL Eppendorf tube. Leave the reaction mixture on ice whilst the outside buffer is prepared in a 10 mL tube. Adjust the pH of the outside buffer using 1 M KOH prior to making up the final volume with water.





The quantities listed in this section are for one 500 L reaction mixture with 5 mL outside buffer. An Excel spreadsheet is available online ([http://rsc.anu.edu.au/~go http://rsc.anu.edu.au/~go]) that facilitates scaling to different volumes, number of reactions or different vector concentrations. The quantities of vectors listed assume stock solutions with a concentration of 1 mg plasmid per mL to achieve final concentrations in the reaction mixture of 16 g/mL, as per Table 1. If different volumes of vector solutions are to be added, adjust the quantity of water so that the final volume of the reaction is 500 L. The pH of the outside buffer must be adjusted to 7.5 using 1 M KOH, and the volume of water added adjusted accordingly. T7 RNA polymerase must be supplied if the expression of the target gene is under control of the T7 promoter. The pKO1166 vector is used in this example rather than purified T7 RNA polymerase so that fresh polymerase is continuously generated during the reaction (22). In most cases, pKO1166 delivers higher protein yields than purified T7 RNA polymerase, whereas experiments with incorporation of non-natural amino acids may depend on provision of the purified polymerase, which should be supplied at a final concentration of 32 g/mL. When using the purified polymerase enzyme there is no need for additional Lalanine to be added to the reaction mixture. The water volume to be added must be adjusted accordingly (21). The synthesis and purification of T7 RNA polymerase from a strain containing pKO1166 has been described in (14).