IGEM:IMPERIAL/2007/CFS/Design/Protocols: Difference between revisions
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**0.05% (v/v) 2-mercaptoethanol (2-ME) | **0.05% (v/v) 2-mercaptoethanol (2-ME) | ||
====Procedure==== | |||
'''Growing the cells''' | '''Growing the cells''' | ||
#Grow ''E. coli'' strain BL21 (DE3) cells at 37°C in 3L of 2xYT medium till O.D.600 = 0.6.'''Ensure vigorous agitation and aeration.''' | #Grow ''E. coli'' strain BL21 (DE3) cells at 37°C in 3L of 2xYT medium till O.D.600 = 0.6.'''Ensure vigorous agitation and aeration.''' | ||
Line 46: | Line 47: | ||
#Centrifuge and weigh the wet cell pellets before storing them at -80°C. | #Centrifuge and weigh the wet cell pellets before storing them at -80°C. | ||
''(Note: The cells may take more than 1 day to grow to O.D.600 = 4.5.)'' | ''(Note: The cells may take more than 1 day to grow to O.D.600 = 4.5.)'' | ||
===Day 2=== | ===Day 2=== | ||
Line 77: | Line 77: | ||
''(Note: For the ATP regenerating system in the pre-incubation solution, phosphoenolpyruvate and pyruvate kinase are used instead of creatine phosphate and creatine kinase. This is due to cost considerations.)'' | ''(Note: For the ATP regenerating system in the pre-incubation solution, phosphoenolpyruvate and pyruvate kinase are used instead of creatine phosphate and creatine kinase. This is due to cost considerations.)'' | ||
====Procedure==== | |||
'''Lysing the cells''' | '''Lysing the cells''' | ||
#Suspend thawed cells in 12.7ml of buffer B per 10g of wet cells. | #Suspend thawed cells in 12.7ml of buffer B per 10g of wet cells. | ||
Line 92: | Line 93: | ||
''(Note: Protease inhibitors are added to pre-incubation solution to prevent degradation of proteins required for gene expression.)'' | ''(Note: Protease inhibitors are added to pre-incubation solution to prevent degradation of proteins required for gene expression.)'' | ||
===Notes=== | ===Notes=== | ||
Line 101: | Line 101: | ||
**The original protocol does not use protease inhibitors in the pre-incubation solution. | **The original protocol does not use protease inhibitors in the pre-incubation solution. | ||
==Protocol for Preparation of ''E. coli'' S12 Extract== | |||
===Day 1=== | |||
====Equipment==== | |||
*37°C shaking incubator | |||
*1L conical flasks x 3 | |||
*Pipette fillers + pipettes (5ml, 10ml and 25ml) | |||
*Spectrometer + cuvettes | |||
*Weighing scale | |||
*Centrifuge + 150ml centrifuge tubes | |||
*Pipettes + pipette tips (20µl, 200µl and 1000µl) | |||
====Reagent==== | |||
*2xYT medium | |||
*IPTG | |||
*Buffer A | |||
**10mM Tris-acetate (pH 8.2) | |||
**14mM Mg-acetate | |||
**60mM K-lutamate | |||
**1mM dithiothreitol (DTT) | |||
**0.05% (v/v) 2-mercaptoethanol (2-ME) | |||
====Procedure==== | |||
'''Growing the cells''' | |||
#Grow ''E. coli'' strain BL21 (DE3) cells at 37°C in 3L of 2xYT medium till O.D.600 = 0.6.'''Ensure vigorous agitation and aeration.''' | |||
#Add 1mM IPTG to cell culture to express T7 RNA polymerase. | |||
#Harvest cells when O.D.600 = 4.5. '''At this point, cells are at mid-log phase.''' | |||
#Wash cells three times by suspending them in 20ml of buffer A per gram of wet cells. | |||
#Centrifuge and weigh the wet cell pellets before storing them at -80°C. | |||
''(Note: The cells may take more than 1 day to grow to O.D.600 = 4.5.)'' | |||
===Day 2=== | |||
====Equipment==== | |||
*Pipette filler + pipettes (5ml, 10ml and 25ml) | |||
*Weighing scale | |||
*French press + French press cell | |||
*Centrifuge + 50ml centrifuge tubes | |||
*Pipette + pipette tips (20µl, 200µl, 1000µl) | |||
*37°C shaking incubator | |||
*Dialysis membrane with molecular weight cut-off of 10,000 | |||
*Magnetic stirrer | |||
*4°C cold room | |||
====Reagent==== | |||
*Buffer B | |||
**10mM Tris-acetate (pH 8.2) | |||
**14mM Mg-acetate | |||
**60mM K-glutamate | |||
**1mM DTT | |||
====Procedure==== | |||
'''Lysing the cells''' | |||
#Suspend thawed cells in 12.7ml of buffer B per 10g of wet cells. | |||
#Disrupt cells in a French press cell at a constant pressure of 20,000psi.'''This is about 140,000kPa.''' | |||
'''Retaining the cell extract''' | |||
#Centrifuge the crude lysate at 12,000RCF for 10min at 4°C. | |||
#Carefully remove the top layer of the supernatant (lipid layer) and the pellet. | |||
#Briefly pre-incubate the recovered supernatant at 37°C for 30min. | |||
#Divide resulting S12 extract into small aliquots and store at -80°C. | |||
===Notes=== | |||
* Total time required: ~ 3 days. | |||
* The protocol is based on [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T3C-4K242VK-1&_user=10&_coverDate=12%2F01%2F2006&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=fbf36c742708c667b5c4481856ca22b5 Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system] by Kim DM et al. | |||
*We did not prepare any ''E. coli'' S12 extract because of the lack of reagents and limited budget. | |||
==Protocol for Vesicle Formation== | |||
===Day 1=== | |||
====Equipment==== | |||
*Nitrogen tap + plastic tubing | |||
*Desiccator connected to a vacuum | |||
*100ml glass bottle | |||
*Sonicator with medium-sized probe | |||
*Ice bath | |||
*25°C incubator | |||
*Pipette + pipette tips (1000µl) | |||
====Reagents==== | |||
* 10ml dodecane | |||
* 12.5µl 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0% | |||
====Procedure==== | |||
'''Preparing the lipid-oil suspension for the inner leaflet''' | |||
#Place 125µl of the 20mg/ml DOPC solution in a 100ml glass bottle. | |||
#With the plastic tubing and 1ml pipette tip, evaporate the chloroform under nitrogen to obtain a dry, thin lipid film. | |||
#Put the bottle in a desiccator connected to a vacuum for 1h. | |||
#Add 50ml of mineral oil to reach a final lipid concentration of 0.05mg/ml. | |||
#Set the sonicator probe to pulse 1, timer at 30mins. | |||
#Put the bottle containing the suspension in the ice bath. | |||
#Secure the sonicator probe inside the bottle, and set the amplitude to a reading of 10 when it is sonicating. | |||
#Sonicate the suspension for 30mins. | |||
#Leave overnight at 25°C to ensure that the lipid molecules are fully dispersed in oil. | |||
===Day 2=== | |||
====Equipment==== | |||
*Magnetic stirrer | |||
*Centrifuge + 1-inch glass centrifuge tubes | |||
*Pipette + pipette tips (200µl, 1000µl) | |||
*50ml glass tube | |||
*5ml syringe | |||
*Long 16-gauge stainless steel needle | |||
====Reagents==== | |||
*10ml ddH2O | |||
*Tris buffer | |||
*NaCl | |||
*Reporter | |||
====Procedure==== | |||
'''Emulsifying the aqueous solution''' (while the interface settles) | |||
#Separate about 5ml of the lipid-oil suspension into a glass container. '''This is for the interface preparation.''' | |||
#Prepare a 10ml solution A with 100mM NaCl and 5 mM Tris buffer at pH 7.4. | |||
#Prepare solution B by adding a suitable quantity of reporter to 1ml of solution A. | |||
#Add 250µl of solution B to the 45ml lipid-oil suspension in mineral oil. | |||
#Gently stir the mixture with a magnetic stir bar for 3h. | |||
'''Preparing the interface''' (to be done while the emulsion is mixing) | |||
#Place 2ml of lipid-oil suspension over 3ml of solution A in a 1-inch-diameter centrifuge tube. | |||
#Leave for 2–3h for lipids to achieve the coverage of the interface surface. | |||
'''Forming the vesicles''' | |||
#Pour 100µl of the inverted emulsion over the interface. | |||
#Centrifuge at 120g for 10min. | |||
'''Collecting the vesicles''' | |||
#Using a 5ml syringe with a long 16-gauge stainless steel needle, collect some of solution A. | |||
#Expel some of the solution to remove all air from the syringe and needle. | |||
#With the tip of the needle in the aqueous phase, gently expel the solution contained in the syringe. | |||
#Gently recirculate the buffer several times. | |||
#Aspirate most of the solution into the syringe, and remove the needle from the solution. | |||
#Wipe the tip of the needle clean. | |||
#Unload the vesicle suspension into its final container. | |||
''(Note: Use optical microscopy to check that the vesicles obtained arenot deformed or aggregated.)'' | |||
===Notes=== | |||
*Time Required: | |||
**The lipid-oil suspension preparation takes about 2h (with a 1h waiting period 15min into the procedure), before being left overnight. | |||
*The remainder of the procedure takes another 4h, with one 2h waiting period after an initial 1h preparation. | |||
**Total working time in the lab is around 3 hours. | |||
* 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 30min. | |||
*Do not use rubber tubing in the nitrogen evaporation. '''This emits 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. | |||
*Use of GFP as a visual signal may require osmolarity considerations. | |||
*The reporter in solution B is optional. '''The vesicles may be visible without it.''' | |||
*The interface should settle for more than 2h, but less than 3h. '''More than 3h causes the lipids to clump.''' | |||
==Protocol for Preparation of ''E. coli'' S12 Extract== | ==Protocol for Preparation of ''E. coli'' S12 Extract== |
Revision as of 02:40, 12 September 2007
Cell-Free Systems
Protocol for Preparation of E. coli S30 Extract
Day 1
Equipment
- 37°C shaking incubator
- 1L conical flasks x 3
- Pipette fillers + pipettes (5ml, 10ml and 25ml)
- Spectrometer + cuvettes
- Weighing scale
- Centrifuge + 150ml centrifuge tubes
- Pipettes + pipette tips (20µl, 200µl and 1000µl)
Reagent
- 2xYT medium
- IPTG
- Buffer A
- 10mM Tris-acetate (pH 8.2)
- 14mM Mg-acetate
- 60mM K-lutamate
- 1mM dithiothreitol (DTT)
- 0.05% (v/v) 2-mercaptoethanol (2-ME)
Procedure
Growing the cells
- Grow E. coli strain BL21 (DE3) cells at 37°C in 3L of 2xYT medium till O.D.600 = 0.6.Ensure vigorous agitation and aeration.
- Add 1mM IPTG to cell culture to express T7 RNA polymerase.
- Harvest cells when O.D.600 = 4.5. At this point, cells are at mid-log phase.
- Wash cells three times by suspending them in 20ml of buffer A per gram of wet cells.
- Centrifuge and weigh the wet cell pellets before storing them at -80°C.
(Note: The cells may take more than 1 day to grow to O.D.600 = 4.5.)
Day 2
Equipment
- Pipette filler + pipettes (5ml, 10ml and 25ml)
- Weighing scale
- French press + French press cell
- Centrifuge + 50ml centrifuge tubes
- Pipette + pipette tips (20µl, 200µl, 1000µl)
- 37°C shaking incubator
- Dialysis membrane with molecular weight cut-off of 10,000
- Magnetic stirrer
- 4°C cold room
Reagent
- Buffer B
- 10mM Tris-acetate (pH 8.2)
- 14mM Mg-acetate
- 60mM K-glutamate
- 1mM DTT
- Pre-incubation solution
- 293.3mM Tris-acetate (pH 8.2)
- 2mM Mg-acetate
- 10.4mM ATP
- 4.4mM DTT
- 0.04mM amino acids
- 16.9mM phosphoenolpyruvate
- 0.77U/ml pyruvate kinase
(Note: For the ATP regenerating system in the pre-incubation solution, phosphoenolpyruvate and pyruvate kinase are used instead of creatine phosphate and creatine kinase. This is due to cost considerations.)
Procedure
Lysing the cells
- Suspend thawed cells in 12.7ml of buffer B per 10g of wet cells.
- Disrupt cells in a French press cell at a constant pressure of 20,000psi.This is about 140,000kPa.
Retaining the cell extract
- Centrifuge the crude lysate at 30,000RCF for 30min at 4°C.
- Carefully remove the top layer of the supernatant (lipid layer) and the pellet and centrifuge again.
- Shake the final supernatant at 100rpm.
- Gradually add 3ml of the pre-incubation solution to 10ml of the supernatant.
- Pre-incubate the supernatant with gentle shaking at 37°C for 80min. This degrades endogenous genetic content (DNA and mRNA).
- Dialyze the pre-incubated sample for 45min each at 4°C against 50 volumes of buffer B using a membrane with molecular weight cut-off of 10,000. Repeat the dialysis step three times.
- Centrifuge the retained extract at 4000RCF for 10min at 4°C to obtain the supernatant.
- Divide resulting S30 extract into small aliquots and store at -80°C.
(Note: Protease inhibitors are added to pre-incubation solution to prevent degradation of proteins required for gene expression.)
Notes
- Total time required: ~ 3 days.
- The protocol is based on Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system by Kim DM et al.
- Modifications to protocol:
- The original protocol uses creatine phosphate and creatine kinase instead of phosphoenolpyruvate and pyruvate kinase.
- The original protocol does not use protease inhibitors in the pre-incubation solution.
Protocol for Preparation of E. coli S12 Extract
Day 1
Equipment
- 37°C shaking incubator
- 1L conical flasks x 3
- Pipette fillers + pipettes (5ml, 10ml and 25ml)
- Spectrometer + cuvettes
- Weighing scale
- Centrifuge + 150ml centrifuge tubes
- Pipettes + pipette tips (20µl, 200µl and 1000µl)
Reagent
- 2xYT medium
- IPTG
- Buffer A
- 10mM Tris-acetate (pH 8.2)
- 14mM Mg-acetate
- 60mM K-lutamate
- 1mM dithiothreitol (DTT)
- 0.05% (v/v) 2-mercaptoethanol (2-ME)
Procedure
Growing the cells
- Grow E. coli strain BL21 (DE3) cells at 37°C in 3L of 2xYT medium till O.D.600 = 0.6.Ensure vigorous agitation and aeration.
- Add 1mM IPTG to cell culture to express T7 RNA polymerase.
- Harvest cells when O.D.600 = 4.5. At this point, cells are at mid-log phase.
- Wash cells three times by suspending them in 20ml of buffer A per gram of wet cells.
- Centrifuge and weigh the wet cell pellets before storing them at -80°C.
(Note: The cells may take more than 1 day to grow to O.D.600 = 4.5.)
Day 2
Equipment
- Pipette filler + pipettes (5ml, 10ml and 25ml)
- Weighing scale
- French press + French press cell
- Centrifuge + 50ml centrifuge tubes
- Pipette + pipette tips (20µl, 200µl, 1000µl)
- 37°C shaking incubator
- Dialysis membrane with molecular weight cut-off of 10,000
- Magnetic stirrer
- 4°C cold room
Reagent
- Buffer B
- 10mM Tris-acetate (pH 8.2)
- 14mM Mg-acetate
- 60mM K-glutamate
- 1mM DTT
Procedure
Lysing the cells
- Suspend thawed cells in 12.7ml of buffer B per 10g of wet cells.
- Disrupt cells in a French press cell at a constant pressure of 20,000psi.This is about 140,000kPa.
Retaining the cell extract
- Centrifuge the crude lysate at 12,000RCF for 10min at 4°C.
- Carefully remove the top layer of the supernatant (lipid layer) and the pellet.
- Briefly pre-incubate the recovered supernatant at 37°C for 30min.
- Divide resulting S12 extract into small aliquots and store at -80°C.
Notes
- Total time required: ~ 3 days.
- The protocol is based on Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system by Kim DM et al.
- We did not prepare any E. coli S12 extract because of the lack of reagents and limited budget.
Protocol for Vesicle Formation
Day 1
Equipment
- Nitrogen tap + plastic tubing
- Desiccator connected to a vacuum
- 100ml glass bottle
- Sonicator with medium-sized probe
- Ice bath
- 25°C incubator
- Pipette + pipette tips (1000µl)
Reagents
- 10ml dodecane
- 12.5µl 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0%
Procedure
Preparing the lipid-oil suspension for the inner leaflet
- Place 125µl of the 20mg/ml DOPC solution in a 100ml glass bottle.
- With the plastic tubing and 1ml pipette tip, evaporate the chloroform under nitrogen to obtain a dry, thin lipid film.
- Put the bottle in a desiccator connected to a vacuum for 1h.
- Add 50ml of mineral oil to reach a final lipid concentration of 0.05mg/ml.
- Set the sonicator probe to pulse 1, timer at 30mins.
- Put the bottle containing the suspension in the ice bath.
- Secure the sonicator probe inside the bottle, and set the amplitude to a reading of 10 when it is sonicating.
- Sonicate the suspension for 30mins.
- Leave overnight at 25°C to ensure that the lipid molecules are fully dispersed in oil.
Day 2
Equipment
- Magnetic stirrer
- Centrifuge + 1-inch glass centrifuge tubes
- Pipette + pipette tips (200µl, 1000µl)
- 50ml glass tube
- 5ml syringe
- Long 16-gauge stainless steel needle
Reagents
- 10ml ddH2O
- Tris buffer
- NaCl
- Reporter
Procedure
Emulsifying the aqueous solution (while the interface settles)
- Separate about 5ml of the lipid-oil suspension into a glass container. This is for the interface preparation.
- Prepare a 10ml solution A with 100mM NaCl and 5 mM Tris buffer at pH 7.4.
- Prepare solution B by adding a suitable quantity of reporter to 1ml of solution A.
- Add 250µl of solution B to the 45ml lipid-oil suspension in mineral oil.
- Gently stir the mixture with a magnetic stir bar for 3h.
Preparing the interface (to be done while the emulsion is mixing)
- Place 2ml of lipid-oil suspension over 3ml of solution A in a 1-inch-diameter centrifuge tube.
- Leave for 2–3h for lipids to achieve the coverage of the interface surface.
Forming the vesicles
- Pour 100µl of the inverted emulsion over the interface.
- Centrifuge at 120g for 10min.
Collecting the vesicles
- Using a 5ml syringe with a long 16-gauge stainless steel needle, collect some of solution A.
- Expel some of the solution to remove all air from the syringe and needle.
- With the tip of the needle in the aqueous phase, gently expel the solution contained in the syringe.
- Gently recirculate the buffer several times.
- Aspirate most of the solution into the syringe, and remove the needle from the solution.
- Wipe the tip of the needle clean.
- Unload the vesicle suspension into its final container.
(Note: Use optical microscopy to check that the vesicles obtained arenot deformed or aggregated.)
Notes
- Time Required:
- The lipid-oil suspension preparation takes about 2h (with a 1h waiting period 15min into the procedure), before being left overnight.
- The remainder of the procedure takes another 4h, with one 2h waiting period after an initial 1h preparation.
- Total working time in the lab is around 3 hours.
- 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 30min.
- Do not use rubber tubing in the nitrogen evaporation. This emits 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.
- Use of GFP as a visual signal may require osmolarity considerations.
- The reporter in solution B is optional. The vesicles may be visible without it.
- The interface should settle for more than 2h, but less than 3h. More than 3h causes the lipids to clump.
Protocol for Preparation of E. coli S12 Extract
Day 1
Equipment
- 37°C shaking incubator
- 1L conical flasks x 3
- Pipette fillers + pipettes (5ml, 10ml and 25ml)
- Spectrometer + cuvettes
- Weighing scale
- Centrifuge + 150ml centrifuge tubes
- Pipettes + pipette tips (20µl, 200µl and 1000µl)
Reagent
- 2xYT medium
- IPTG
- Buffer A
- 10mM Tris-acetate (pH 8.2)
- 14mM Mg-acetate
- 60mM K-lutamate
- 1mM dithiothreitol (DTT)
- 0.05% (v/v) 2-mercaptoethanol (2-ME)
Growing the cells
- Grow E. coli strain BL21 (DE3) cells at 37°C in 3L of 2xYT medium till O.D.600 = 0.6.Ensure vigorous agitation and aeration.
- Add 1mM IPTG to cell culture to express T7 RNA polymerase.
- Harvest cells when O.D.600 = 4.5. At this point, cells are at mid-log phase.
- Wash cells three times by suspending them in 20ml of buffer A per gram of wet cells.
- Centrifuge and weigh the wet cell pellets before storing them at -80°C.
(Note: The cells may take more than 1 day to grow to O.D.600 = 4.5.)
Day 2
Equipment
- Pipette filler + pipettes (5ml, 10ml and 25ml)
- Weighing scale
- French press + French press cell
- Centrifuge + 50ml centrifuge tubes
- Pipette + pipette tips (20µl, 200µl, 1000µl)
- 37°C shaking incubator
- Dialysis membrane with molecular weight cut-off of 10,000
- Magnetic stirrer
- 4°C cold room
Reagent
- Buffer B
- 10mM Tris-acetate (pH 8.2)
- 14mM Mg-acetate
- 60mM K-glutamate
- 1mM DTT
Lysing the cells
- Suspend thawed cells in 12.7ml of buffer B per 10g of wet cells.
- Disrupt cells in a French press cell at a constant pressure of 20,000psi.This is about 140,000kPa.
Retaining the cell extract
- Centrifuge the crude lysate at 12,000RCF for 10min at 4°C.
- Carefully remove the top layer of the supernatant (lipid layer) and the pellet.
- Briefly pre-incubate the recovered supernatant at 37°C for 30min.
- Divide resulting S12 extract into small aliquots and store at -80°C.
Notes
- Total time required: ~ 3 days.
- The protocol is based on Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system by Kim DM et al.
- We did not prepare any E. coli S12 extract because of the lack of reagents and limited budget.