TITLE: Production of cellular reagents using IPTG
AUTHORS: Shalo  Minette, Jenny  Molloy, Nadine             Mowoh, Stephane  Fadanka

[DESCRIPTION]
This protocol documents the production of cellular reagents. 
Cellular reagents are defined as common molecular biology enzymes expressed in E.coli but not subsequently purified before use i.e. dried E.coli cells are used as the reagent (Bhadra et al (2018)).

This protocol provides instructions for IPTG (T7 based) expression.

[BEFORE_START]
Ensure all materials and reagents needed are available and all culture media prepared.

[GUIDELINES]
This protocol describes the use of IPTG in inducing protein expression in E.coli BL 21 DE3 strain.

[STEPS]
SECTION: Preparation of overnight starter culture
1. 
 
From glycerol stock

Remove the glycerol stock from -20°C.
Open the tube and use a sterile loop, sterile toothpick or sterile pipette tip to scrape some of the frozen bacteria off the top. Do not let the glycerol stock thaw!
Streak onto LB agar plate which contains appropriate antibiotic, label with full details.
Return glycerol stock to freezer.
Incubate plate at37 °C  in a static incubator
Next day, check plate for growth of bacteria.
Prepare 5 mL LB broth plus appropriate antibiotic as described in this protocol. 

e.g. 2.5µl 100 mg/mL Kanamycin stock in 5 mL LB broth, to give 50 µg/mL final kanamycin concentration.

Inoculate prepared 5 mL LB Broth + Antibiotic in a 50 mL Falcon tube with 1 E.coli colony from the overnight the plate.
Label and place the falcon tube in a shaking incubator at 37 °C  
Store plate with colonies on at +4°C for up to 3 weeks.


 From a pre-streaked plate

Remove the plate from +4°C
Use a wire loop to pick a colony from the plate
Inoculate a prepared 5 mL LB Broth + Antibiotic in a 50 mL Falcon tube with 1 E.coli colony from the plate
Label and place the falcon tube in shaking incubator at 37 °C 
Store plate with colonies on at +4°C for up to 3 weeks.
      

SECTION: Growth of main culture and preparation of cellular reagents
2. Growing main culture for IPTG-protein expression

Prepare 50 mL LB broth plus appropriate antibiotic in a 100 mL flask
Inoculate the 50 mL LB Broth + Antibiotic with 0.5 % volume of the starter culture 
e.g.250 µL of the overnight culture in 50 mL LB + Antibiotic 
Grow at   until A600 = 0.5 
Induce  expression by adding0.5 millimolar (mM) IPTG final concentration 

Calculate the amount of IPTG as follows:

Amount of IPTG to add (µL) = concentration IPTG sought (mM) x volume of culture (µL)
                                                                  Stock IPTG conc (mM)

e.g.31.25 µL of 0.8 Molarity (M) stock to 50 mL

Continue to incubate for  .
Decant 1.5 mL (or desired volume depending on the tubes available) into centrifuge tubes or Eppendorf tubes.
Centrifuge at 4000 rpm, 15 min , pour off and discard the supernatant and keep the pellets at 4 °C.
Now make another set of tubes: This set is to be used for SDS PAGE to confirm successful protein expression.

    
After confirming successful protein expression
Wash cells

Remove from the fridge the first set of tubes and resuspend cell pellet into 1.5 mL of cold PBS (vol of PBS = vol of harvested cell) – First wash

e.g. 1.5 mL harvested resuspend in 1.5 mL cold 1X PBS

Centrifuge at 4000 rpm, 15 min 
Perform second wash 
Resuspend pellet into 1.5 mL of cold 1X PBS
SECTION: Growth of main culture and preparation of cellular reagents
3. Aliquoting
Measure A600 of a neat, 1:10 or 1:100 dilution. Multiply the value to get the actual final A600 number.

Calculate the volume of your final cell suspension that would contain 2 x 108 cells, using the equation:  

volume containing 2x108 cells = 200/final A600 of cell suspension. 

e.g. if your final A600 is 6.5, then

volume containing 2x108 cells = 200/6.5
volume containing 2x108 cells = 31µL
 

Aliquot either single reaction or 10X reactions worth of cellular reagents into 8-tube strips of 0.2 mL PCR tubes.

e.g. using the example above, 3.1 µL (1x reaction) or 31 µL (10x).
 

SECTION: Growth of main culture and preparation of cellular reagents
4. Heating to kill the bacteria 

Transfer the 0.2 mL PCR tubes into a thermocycler 
Set a program for heating on the thermocycler at 60 °C for 10 min (leave some tubes unheated to serve as control and label the tubes accordingly).
 

SECTION: Growth of main culture and preparation of cellular reagents
5. Drying

Leave tube lids open
Place the tube strips with pre- heated aliquoted cellular reagents carefully in a vacuum tupperware 1/2 filled with desiccant.
place the lid of the tupperware on top and close gently making sure it closes properly on all the sides
Use the vacuum pump to create a vacuum in the tupperware by pumping several times until the lid is visibly tough and sunken. 
Place the container static incubator 5 min at 37 °C
After 18-24 hours check to see if the cellular reagents are completely dry. 
Once dry, close the lids and place in a small bag at 4 °C with a small amount of desiccant.

 
      
SECTION: Growth of main culture and preparation of cellular reagents
6. 
Check the effectiveness of the heating step

Reconstitute one of the pre-heated tubes with 30 µLPCR water 
Mix by gently tapping onto the bottom of the tube and allow to stand for 5 min
Use a micropipette to aliquot 10 µLinoculate on an LB agar plate supplemented with Kanamycin (100 mg/mL)
Use a spreader to evenly spread the inoculum across the surface of the plate
Repeat the above steps but this time reconstituting the control tube (tubes where cells were not pre-heated)
Place both plates in a static incubator and incubated at 37 °C5 min

Check plates: 
The plate from pre-heated tubes should show no growth of bacteria confirming the complete killing of bacteria cells in cellular reagents making them safer to handle.
The plate from non-heated tubes should show visible growth of bacteria confirming the need to preheat cellular reagents at 60c for 10 mins before drying and subsequent use.

SECTION: Quality control
7. Proceed to carry out quality control of this batch of cellular reagents by testing the functionality and assessing the them for endonuclease activity.
After passing the quality control test, the cellular reagents can be used in a PCR amplification reaction.