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

=In-Veso Gene Expression: Design - Experimental Protocols=

 Introduction Specifications Design Modelling Implementation Testing/Validation Notes References </ul> <br style="clear:both">

S30 E.coli Extract Protocol
The protocol is based on that of Karp and Pellinen.

Chemicals and reagents
From [[media:S30.gif|preparation of S30 extract by Pellinen Pellinen ]] 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

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).

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

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

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.

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.