TITLE: Generation of E. coli MG1655 whole cell lysate for proteomics applications
AUTHORS: David Butcher

[DESCRIPTION]
This protocol describes the culture of E. coli MG1655 from a glycerol stock solution and subsequent lysis. It is intended for generating small quantities of whole cell lysate from cells grown in nutrient-rich (e.g. LB or TB medium) and/or nutrient-poor (e.g. M9 minimal) media for proteomics analysis. Culture and lysis of cells in a single Bio-Reaction tube minimizes the possibility of contamination.

All cell culture steps should be carried out using sterile materials and proper aseptic technique. This procedure is intended to be carried out in a biosafety cabinet, but it can also easily be performed on a bench top with a Bunsen burner.

Applications of this protocol at the National MagLab have utilized E. coli stocks from the Coli Genetic Stock Center at Yale University. The final lysate typically contains 300 - 500 μg/mL of protein for M9 minimal medium cultures and 750 - 1000 μg/mL for LB medium cultures, as determined by BCA assay.

The cell lysis protocol is adapted from Preparing human WCL for analysis by qualitative TDMS by Caroline DeHart, with a few changes.

[STEPS]
SECTION: Cell culture
10. Seal lids of Bio-Reaction tubes with Parafilm (or swap for lids without pores) and freeze cell pellets at -80 °C until ready to perform lysis (or at least one hour).

SECTION: Cell culture
9. Centrifuge tubes at 4000 x g, 15 min, 4 °C, then decant culture medium from each tube leaving pellet behind.
 


SECTION: Cell culture
7. Incubate tubes in an orbital shaker ( ). Use UV-vis spectrophotometer to monitor optical density at 600 nm (OD600) and plot growth curve. 
 

SECTION: Cell culture
6. Inoculate each Bio-Reaction tube with 50 µL (1/300th volume) of the saturated culture grown overnight.
 

SECTION: Cell culture
2. Add 5 mL of liquid media to sterilized 16 x 150 mm culture tubes. 
 

SECTION: Cell culture
1. Streak E. coli sample(s) on non-selective LB and/or M9 minimal media agar plates. Incubate at 37 °C until colonies are visible and well separated. 
 


SECTION: Cell lysis
11. Begin cooling the centrifuge to 4 °C.

SECTION: Cell lysis
13. Thaw cell pellets on ice for 15 mins, then resuspend each pellet in chilled lysis buffer. Use 1 mL of lysis buffer per 5 mL of suspension culture.
SECTION: Cell lysis
14. Incubate lysate on ice for 20 mins. Swirl to mix every 5 min.
SECTION: Cell lysis
15. Add 1 µL of 1 M MgCl2 per 1 mL of lysis buffer to the lysate, to a final concentration of 1 mM.
SECTION: Cell lysis
16. Add nuclease to digest genomic DNA.

If using Benzonase nuclease HC:

Add 0.5 µL (125 U) of Benzonase nuclease HC for each mL of lysate. Pipette to ensure complete mixing.

If using DNAse I:

Add 10 µL (20 U) of New England Biolabs DNase I for each mL of lysate. Pipette to ensure complete mixing.

If using sonication:

It is acceptable to rely on a subsequent sonication step to break down genomic DNA and skip this step. It may be necessary to increase the number of sonication cycles to reduce lysate viscosity.
SECTION: Cell lysis
18. Pellet cellular debris by centrifugation at 12,500 G for 20 mins at 4 °C.
 

SECTION: Cell lysis
16.1. Incubate lysate at 37 °C for 20 min. Invert to mix every 5 mins.
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16.2. Chill lysates on ice for 5 mins.
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19. Determine total protein concentration for each lysate by performing a Bicinchoninic acid (BCA) or other appropriate assay.
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17. Optional sonication step

Place tube containing cell pellet resuspended in lysis buffer on ice. Set sonicator power to lowest setting and place probe tip close to the bottom of tube without touching. Turn on sonicator and adjust power until the surface of the buffer is agitated, but not foaming. Pulse for five cycles of 10 seconds followed by 30 seconds of rest.
SECTION: Cell lysis
12. Prepare fresh lysis buffer according to the following table and chill on ice. This recipe produces 10 mL of lysis buffer, which is enough for cells from 45 mL of suspension culture. Scale up or down as needed.

 

 

SECTION: Cell lysis
20. Aliquot lysates into smaller portions appropriate to downstream applications (e.g. 50 or 100 μg for proteomics analysis). Store aliquots at -80 ºC until ready to use. 
 



SECTION: Cell culture
4. Incubate culture tubes in an orbital shaker ( ) overnight (approximately 960 min). 
 


SECTION: Cell culture
3. Inoculate each culture tube with E. coli cells taken from a single colony on each agar plate.
 

SECTION: Cell culture
5. Add 15 mL of sterile liquid media to 50 mL Bio-Reaction tubes.
 

SECTION: Cell culture
8. Grow cells to mid-to-late exponential stage, with OD600 of approximately 1.0.