TITLE: Plate Protein Expression on Autoinduction media
AUTHORS: Stephane Fadanka, Chiara Gandini

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The current protocol describes the preparation and use of 2X YT</span><span style = "font-weight:bold;"> </span><span>autoinduction medium for recombinant protein expression on Petri dishes. This protocol allows for reproducible and time effective expression experiments to be undertaken with minimal user intervention as compared to standard procedures using IPTG.</span></div><br/><br/></div>

[STEPS]
?. [Preparation of the Overnight Pre-inoculum]
Grow culture   of desired bacteria strain in LB broth supplemented with the appropriate antibiotic, grow overnight at 37C .
10 mL
37 °C
?. [Preparation of Auto-induction media]
Prepare the needed amount of 4xYT autoinduction agar medium
?. [Preparation of Auto-induction media]
Composition: 4x YT (0.5L in 1L bottle) liquid6g Na2HPO43gKH2PO420gTryptone5g Yeast Extract5gNaCl
?. [Preparation of Auto-induction media]
Weigh and dissolve all powders and salts in  of distilled water and transfer to a Duran bottle.
0.5 L
1 L
?. [Preparation of Auto-induction media]
Prepare 50% glycerol, 10% glucose and 5% lactose solutions for reconstitution of the autoinduction media after sterilisation:
?. [Preparation of Auto-induction media]
50% (vol/vol) glycerol- Measure 50ml of glycerol in a 250ml bottle- Add 50 ml of distilled water and mix by shaking.
?. [Preparation of Auto-induction media]
10% (weight/volume) glucose- Weight 10g of glucose- Dissolve in 100ml of water
?. [Preparation of Auto-induction media]
5% lactose- Weight 10g of lactose- Dissolve in 200ml of water
?. [Preparation of Auto-induction media]
Enough distilled water to dilute and reconstitute media after sterilisation ()
1 L
?. [Preparation of Auto-induction media]
Sterilize all solutions by autoclaving.
?. [Preparation of Auto-induction media]
After Autoclaving, reconstitute autoinduction medium
?. [Preparation of Auto-induction media]
Reconstitution:Add into the 1L bottle containing 0.5L of 4xYT:   of 50% glycerol  of 10% glucose  of 5% lactose.Bring to 1L with sterile water and carefully shake the flask to mix the solution
12 mL
5 mL
40 mL
?. [Preparation of Auto-induction media]
After reconstituting the media, add an appropriate antibiotic and pour  of reconstituted media in 9cm Petri dishes.
20 mL
?. [Preparation of Auto-induction media]
Allow the plates to solidify for about
?. [Preparation of Auto-induction media]
Inoculate the plates with  of prepared overnight culture using the spreading method.
0.2 mL
?. [Preparation of Auto-induction media]
Incubate the plate overnight at .
37 °C
?. [Collection of Cell Biomass]
Cell Biomass can be collected and stored in a suitable container (1.5ml Eppendorf tubes; 15-50ml Falcon tubes.. etc.) and stored at  or .
-20 °C
-80 °C
?. [Functionality Testing of Expressed Protein ]
Successful expression and functionality of the produced enzyme were evaluated the next day by SDS PAGE and PCR.use a small fraction of stored cell pellet to run an SDS PAGE and check if the protein were successfully expressed:following results were obtained using NuPage Pre-cast gel, MOPs sample Buffer and 2X SDS sample preparation buffer.
?. [Functionality Testing of Expressed Protein ]
The functionality of produced enzymes was assessed for OpenVent DNA Polymerase by carrying out a PCR  reaction using cellular reagents preparations produced with an in-house enzyme: Preparation of Cellular reagents from Plate Auto-induction media            1. Transfert a small lump of cell biomass into a fresh tube and freeze the rest at -20C.           2. Resuspend the cells into 1.2ml of Cold PBS and follow the original protocol for cellular reagents preparation from step6.2.9 (Protocol here).            3. Dilute resuspended cell pellet into cold PBS to obtain a suspension of A600 between 6.5 and 8                 Measure A600 of a neat, 1:10 or 1:100 dilution. Multiply the value to get the actual final A600 number.                 You might have to dilute several times before you get the right OD.            4. Calculate the volume of your final cell suspension to aliquot in each PCR tube                 (that would contain 2 x 108 cells), using the equation:                      Volume to Aliquot = 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             5. 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);             6. Label tubes with reagent, date and operator             7. Incubate the tubes at 60C for 10min in a thermocycler or Heat block (heat treatment)                  to make sure that produced cellular reagents are free from any living bacteria.             8. Place the tube strips with aliquoted cellular reagents carefully in a container 1/2 filled with desiccant,                  leave tubes opened (using vacuum Tupperware is ideal).             9. Place the container overnight in a 37 °C static incubator.             10. After 18-24hrs check to see if the cellular reagents are completely dry.                    Note: Leaving the reagents longer at 37 °C should not hurt their efficacy.             11. Once dry, close the lids and place them in a small bag at +4°C with a small amount of desiccant.
?. [Functionality Testing of Expressed Protein ]
A PCR  reaction was carried out using prepared cellular reagents for OpenVent DNA Polymerases using Lambda genome template 0.5kb. PCR conditions:T annealing 62C, extension 72C for 45s, 37 cycles, 25ul total volume using 1ul of OpenVent, template:lambda genome 1ul of a 1:10 dilution (50ng/ul  concentration of template), primers to amplify 0.5 kb.PCR Results were visualised via Agarose gel electrophoresis on 1.5% agarose gel using TBE buffer system, with 9ul of each amplicon loaded;Bands of expected sizes were spotted after running the gel.