IGEM:IMPERIAL/2007/Projects/In-Veso/Design/Protocols

In-Veso Gene Expression: Design - Experimental Protocols

• Introduction
• Specifications
• Design
• Modelling
• Implementation
• Testing/Validation
• Notes
• References

Preparations

S30 E.coli Extract Protocol

The protocol is based on that of Karp and Pellinen.

Materials

Equipment

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Chemicals and reagents

From preparation of S30 extract by Pellinen [1]
Buffer A

1. 10 mM Tris–acetate buffer (pH 8.2)
2. 14 mM magnesium acetate
3. 60 mM potassium glutamate
4. 1 mM dithiothreitol (DTT) containing 0.05% (v/v) 2-mercaptoethanol (2-ME)

Buffer B

1. 10 mM Tris–acetate buffer (pH 8.2)
2. 14 mM magnesium acetate
3. 60 mM potassium glutamate
4. 1 mM dithiothreitol (DTT)

Pre-incubation solution

1. 293.3 mM Tris–acetate pH 8.2
2. 2 mM magnesium acetate
3. 10.4 mM ATP
4. 200 mM creatine phosphate
5. 4.4 mM DTT
6. 0.04 mM amino acids
7. 26.7 μg/mL creatine kinase

Supplies

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Procedure

1. The cell extracts were prepared from E. coli strain BL21 (DE3) (Novagen, Madison, WI).
2. Grow cells at 37 °C in 3 L of 2xYT medium with vigorous agitation and aeration till OD600=0.6.
3. Add isopropyl-thiogalactopyranoside (IPTG, 1 mM) to the cell culture media to express T7 RNA polymerase.
4. Harvest cells in the mid-log phase (OD600 ≈ 4.5).
5. Wash three times by suspending them in 20 mL of buffer A per gram of wet cells.
6. Centrifuge.
7. Before storing the pellets at −80 °C, weigh the wet cell pellets.
8. Suspend thawed cells (10 g) in 12.7 mL of buffer B.
9. Disrupt cells in a French press cell (Aminco) at a constant pressure of 20,000 psi.
10. Store the pellets at −80 °C.
11. Centrifuge the lysate at 30,000 RCF for 30 min at 4 °C.
12. Carefully removed the top layer of the supernatant (lipid layer) and pellet, and centrifuge them again.
13. Shake the final supernatant at 100 rpm.
14. Gradually add 3 mL of the pre-incubation solution to 10 mL of the supernatant.
15. Incubate supernatant with gentle shaking at 37 °C for 80 min.
16. Dialyze x 4 the pre-incubated sample for 45 min each at 4 °C against 50 volumes of buffer B using a Pierce membrane (SnakeSkin™ Pleated Dialysis Tubing, Rockford, USA) with a molecular weight cut off (MWCO) of 10,000
17. The retained extract was centrifuged at 4000 RCF for 10 min at 4 °C to obtain the supernatant (the S30 extract).

S12 E.coli Extract Protocol

Materials

Equipment

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Chemicals and reagents

• Buffer A
  • 10 mM Tris–acetate buffer (pH 8.2)
  • 14 mM magnesium acetate
  • 60 mM potassium glutamate
  • 1 mM dithiothreitol (DTT) containing 0.05% (v/v) 2-mercaptoethanol (2-ME)
• Buffer B
  • Buffer A without 2-ME

Supplies

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Procedure

1. The cell extracts were prepared from E. coli strain BL21 (DE3) (Novagen, Madison, WI).
2. Grow cells at 37 °C in 3 L of 2xYT medium with vigorous agitation and aeration till OD600=0.6.
3. Add isopropyl-thiogalactopyranoside (IPTG, 1 mM) to the cell culture media to express T7 RNA polymerase.
4. Harvest cells in the mid-log phase (OD600 ≈ 4.5).
5. Wash three times by suspending them in 20 mL of buffer A per gram of wet cells.
6. Centrifuge.
7. Before storing the pellets at −80 °C, weigh the wet cell pellets.
8. Suspend thawed cells (10 g) in 12.7 mL of buffer B
9. Disrupt cells in a French press cell (Aminco) at a constant pressure of 20,000 psi.
10. Store the pellets at −80 °C.
11. Centrifuge crude lysate at 12,000 RCF for 10 min.
12. Briefly incubate recovered supernatant at 37 °C.
13. Divide resulting extract into small aliquots and stored at −80 °C before its use for cell-free protein synthesis

Notes

• S12 cost approximately 50% less than S30.
• S12 shows less batch to batch variation.
• S12 has a substantially higher amount of translational activity than the S30 extract in the Rosetta (DE3) (Novagen)and BL21-Star strains.
• The only exception is that S12 extract for A19 E.coli strain shows much less activity.
• S12 provides approximately 1.5 times higher productivity.
• S12 extract can be prepared with 20% of the cost required for the preparation of the S30 extract
• Preparation time for the S12 extract is only 40% of that required for the S30 extract.

Empty Vesicle Preparation by the Mineral Oil Method

The protocol is based on Engineering Asymmetric Vesicles by Sophie Pautot, Barbara J. Frisken, and D. A. Weitz.

Materials

Equipment

• Sonicator with medium-sized probe (??)
• Nitrogen tap
• Desiccator connected to a vacuum
• 25°C incubator
• Magnetic stirrer
• 120 x g centrifuge (1-inch tubes)
• 200µl pipette
• 1000µl pipette
• 50ml glass tube
• 100ml glass bottle

Chemicals and reagents

• 10ml dodecane
• 12.5µl 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0%
• 10ml ddH2O
• Tris buffer
• NaCl
• Cell extract (Determine quantity)

Supplies

• 1-inch diameter glass centrifuge tube
• 5-ml syringe
• long 16-gauge stainless steel needle
• Ice bath
• 1ml pipette tip
• Plastic tubing

Procedure

Preparing the lipid-oil suspension for the inner leaflet:

1. Place 125 µl of the 20 mg/ml DOPC solution in a 100-ml glass bottle
2. With the plastic tubing and 1ml pipette tip, evaporate the chloroform under nitrogen to obtain a dry, thin lipid film.
3. Put the bottle in a desiccator connected to a vacuum for 1hr
4. Add 50 ml of mineral oil to reach a final lipid concentration of 0.05 mg/ml
5. Set the sonicator probe to pulse 1, timer at 30mins
6. Put the bottle containing the suspension in the ice bath
7. Secure the sonicator probe inside the bottle, and set the amplitude to a reading of 10 when it is sonicating
8. Sonicate the suspension for 30mins
9. Leave overnight at 25°C to ensure that the lipid molecules are fully dispersed in oil

Emulsifying the acqueous solution: (while the interface settles)

1. Separate about 5 ml of the lipid-oil suspension into a glass container (for the interface preparation)
2. Prepare a 10ml solution A with 100 mM NaCl and 5 mM Tris buffer at pH 7.4
3. Prepare solution B by ADDING REPORTER (Determine quantity) to 1ml of solution A.
4. Add 250 µl of solution B to the 45ml lipid-oil suspension in mineral oil
5. Gently stir the mixture with a magnetic stir bar for 3 hours

Preparing the interface: (to be done while the emulsion is mixed)

1. Place 2 ml of lipid-oil suspension over 3 ml of solution A in a 1-inch-diameter centrifuge tube.
2. Leave for 2–3 h for lipids to achieve the coverage of the interface surface

Forming the vesicles:

1. Pour 100 µl of the inverted emulsion over the interface
2. Centrifuge at 120 x g for 10 min

Collecting the vesicles:

1. Using a 5-ml syringe with a long 16-gauge stainless steel needle, collect some of solution A
2. Expel some of the solution to remove all air from the syringe and needle
3. With the tip of the needle in the aqueous phase, gently expel the solution contained in the syringe
4. Gently recirculate the buffer several times
5. Aspirate most of the solution into the syringe, and remove the needle from the solution
6. Wipe the tip of the needle clean
7. Unload the vesicle suspension into its final container

Use optical microscopy to check that the vesicles obtained were not deformed or aggregated.

Time Required

• The lipid-oil suspension preparation takes about 2 hours, before being left overnight (with a 1hr waiting period 15min into the procedure).
• The remainder of the procedure takes another 4 hours, with one 2hr waiting period after an initial 1hr preparation.
• Total working time in the lab is around 3 hours.

Notes

• The original protocol uses anhydrous 99:1 dodecane:silicone oil solution instead of mineral oil
• The original protocol uses POPC instead of DOPC phospholipids
• The original protocol sonicates the suspension in a cleaning sonic bath for 30 min
• Do not use rubber tubing in the nitrogen evaporation - these emit debris into the lipids
• This procedure should form around 10^9 vesicles with 1µm diameter
• Use of salt in the solution A preparation may require osmolarity considerations
• The reporter in solution B is optional - the vesiles may be visible without it
• Use of GFP as a visual signal may require osmolarity considerations
• The interface should settle for more than 2 hours, but less than 3 - more than three and the lipids begin to clump.