Apr 22, 2022
Version 3
Plate Protein Expression on Autoinduction media V.3
- Stephane Fadanka1,
- Chiara Gandini2
- 1MboaLab;
- 2University of Cambridge
Protocol Citation: Stephane Fadanka, Chiara Gandini 2022. Plate Protein Expression on Autoinduction media. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g78n91lwz/v3Version created by Jennifer Molloy
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: April 21, 2022
Last Modified: April 22, 2022
Protocol Integer ID: 61149
Keywords: Plate, Expression, Protein, Auto-intduction
Abstract
The current protocol describes the preparation and use of 2X YT 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.
Materials
Reagents:
- Na2HPO4
- KH2PO4
- Tryptone
- Yeast Extract
- NaCl
- Distilled H2O
- Agar (Bacteriological grade)
- Glycerol
- Glucose
- Lactose
- LB broth supplemented with appropriate antibiotic
- Desired bacteria strain
Equipment:
- 3x250 ml Duran Bottles
- 1L Duran Bottle
- Petri Dishes
- 1.5 ml Eppendorf tubes
- Glas slides
- Autoclave or pressure cooker
- Beakers
- Measuring cylinders
- Electronic scales
- Magnetic stirrer
- Weighing boats
- Laboratory spatula
- Cell spreaders (Glass or plastic)
- 37 C Incubator
- Bunsen Burner
Preparation of the Overnight Pre-inoculum
Preparation of the Overnight Pre-inoculum
Grow culture of desired bacteria strain from glycerol stock or a single colony in 10 mL LB broth supplemented with the appropriate antibiotic, grow overnight in an incubator shaking at 225 rpm, 37°C .
Preparation of Auto-induction media
Preparation of Auto-induction media
30m
30m
Prepare the needed amount of 4xYT autoinduction agar medium
- (It is recommended to prepare and use Auto induction media the same day, make sure to prepare just the amount needed for the experiment) e.g 20ml of Autoinduction in 9cm Petri plates).
Composition of 4x YT autoinduction media for 1L final volume :
6g - Na2HPO4
3g - KH2PO4
20g - Tryptone
5g - Yeast Extract
5g - NaCl
15g - Agar
Note
Note that 1L of media is very difficult to heat to a molten state, and is difficult to fit in a standard microwave or small pressure cooker or autoclave. You may therefore find it more convenient to make media in batches of 500 ml or less. This will provide approx 25 x 90 mm petri dishes.
Weigh and dissolve all powders and salts (including Agar) in 0.5 L of distilled water and transfer to a1 L Duran bottle.
Prepare 50% glycerol, 10% glucose and 5% lactose stock solutions to be used for reconstitution of the autoinduction media after sterilisation:
50% (vol/vol) glycerol
- - Measure 50ml of glycerol in a 250ml bottle
- - Add 50 ml of distilled water and mix by shaking.
10% (weight/volume) glucose
- - Weight 10g of glucose
- - Dissolve in 100ml of water
5% lactose
- - Weight 10g of lactose
- - Dissolve in 200ml of water
Include enough distilled water (500 mL ) to reconstitute autoinduction medium to 1 L after sterilisation.
Sterilize all solutions by autoclaving.
- (Also sterilise microscope slides, as well as Petri Dishes and Cell spreaders if made of glass).
- After Autoclaving, add 50% glycerol, 10% glucose and 5% lactose to the 4xYT to reconstitute the autoinduction medium
Reconstitution of Autoinduction media
Reconstitution of Autoinduction media
30m
30m
- Add into the 1L bottle containing 0.5L of 4xYT: 12 mL of 50% glycerol 5 mL of 10% glucose 40 mL of 5% lactose.
- Sterile pipette tips and containers (such as 50ml Falcon tubes) can be used to accurately measure the exact volumes of the various solutions to reconstitute the medium.
Bring to 1L with sterile water and carefully shake the flask to mix the solution.
Preparation of auto-induction media (4xYT agar)
1. Gather all reagents needed; 2. Mix all ingredients in 1L Duran bottle; 3. Autoclave and Reconstitue 4xYT media
After reconstituting the culture medium, add an appropriate amount of desired antibiotic 50ug/ml Kanamycin and pour 20 mL of reconstituted media in 9cm Petri dishes.
- (Carefully determine the number of plates needed and prepare the volume of media to prepare accordingly make sure to prepare 3 plates per culture and to include replicates for the control as well).
Allow the plates to solidify for about00:30:00
30m
Inoculate the plates with 0.2 mL of prepared overnight culture using the cell spreading method.
Incubate the plate overnight at 37 °C .
Collection of Cell Biomass
Collection of Cell Biomass
- After incubation, period, check cell growth and collect Biomass from each plate using a sterile microscope slide, a scalpel blade or any other available utensil enabling to scrap the surface of the plate without carrying the gel.
- Cell Biomass can be collected and stored in a suitable container (1.5ml Eppendorf tubes; 15-50ml Falcon tubes.. etc.) and stored at -20 °C or -80 °C .
Checking for successful protein expression
Checking for successful protein expression
Successful expression of the produced enzyme was evaluated the next day by SDS PAGE.
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.
- Various enzymes were expressed using prepared auto-induction media and successful enzyme expression was confirmed by the presence of bands at the expected size on the gel.
SDS PAGE gel after enzymes expression using plate auto-induction medium.
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
Functionality testing
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:
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 parameter (MiniPCR mini 16)
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.
Functionality testing of in-house OpenVent DNA polymerase prepared using auto induction media on Plate; 1.5% Agarose gel, 9ul of each PCR template on TBE Buffer system: MW= Molecular weight marker; S= single PCR reaction (2 replicates; -ve= Negative control.