Jun 04, 2025
Efficient Preparation of Competent Cells and Recombinant Protein Expression in E. coli BL21 (DE3) Using IPTG Induction
- 1Parul University
- Durvesh Smita. Burhade: SCIENCE IS ELEGANT
- Durvesh Burhade Lab
Protocol Citation: Durvesh Smita. Burhade 2025. Efficient Preparation of Competent Cells and Recombinant Protein Expression in E. coli BL21 (DE3) Using IPTG Induction. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3wzzqv25/v1
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: In development
We are still developing and optimizing this protocol
Created: June 02, 2025
Last Modified: June 04, 2025
Protocol Integer ID: 219368
Keywords: BL21 (DE3), Competent Cells, Tranformation, Induction & Protein Expression Screening
Abstract
Reproducible method for preparing TSS-competent E. coli BL21 (DE3) cells, transforming them with plasmids, inducing protein expression using IPTG and expression analysis by SDS-PAGE.
Competent Cells Preparation by TSS Method (DAY 1)
Competent Cells Preparation by TSS Method (DAY 1)
Selection of a healthy strain of BL21 (DE3) E. coli Culture from an Agar Plate
Inoculate the isolated colony and incubate
200 rpm, 37°C, 24:00:00 Overnight
Competent Cells Preparation by TSS Method (DAY 2)
Competent Cells Preparation by TSS Method (DAY 2)
40m
40m
Prepare 50 mL of L.B Agar and autoclaved it at 121 °C for 15 Minutes
2 g of L.B Agar in 50 mL Water
Prepare 100 mL of L.B Broth
2.5 g in 100 mL of Water
Dilute the overnight culture in a 1:100 ratio
1 mL of overnight culture in 99 mL Autoclaved L.B Broth
Incubate at 180 rpm, 37°C Till it reaches 0.5 - 0.6 OD
Prepare the TSS Solution (10 mL Total Volume)
10 % of PEG for a 10 mL solution is 1 g
5 % of DMSO for a 10 mL solution is 500 µL
50 mM MgCl2 for a 10 mL solution is 47 mg
Add all the components in 8.45 g of Autoclaved L.B Broth to make total volume of 10 mL
After reaching 0.5 OD, cells should be arrested by incubating the culture on ice for 00:30:00 (30 Minutes)
30m
Cells were harvested by centrifuging at 4000 rpm, 4°C, 00:10:00 , (10 Minutes)
10m
Discard the supernatant. Suspend the cells by adding 1 mL of TSS Solution by tapping them to ice
After resuspension of cells, Aliquote 50 µL in each 1.5 mL Eppendorf tubes (Pre-Chilled)
Checking & Validation (DAY 2)
Checking & Validation (DAY 2)
To check the experiment, add 50 µL of competent cells in 25 ml Agar Plates + Kanamycin Antibiotics
In this step, if we can't see any colonies, it is likely expected that competent cells are ready.
Transformation (DAY 3)
Transformation (DAY 3)
1d 1h 30m
1d 1h 30m
Make 50 mL L.B Agar and pour it into the plates (25 mL each)
In the upcoming steps, this L.B Media will be used
Make 25 mL L.B Broth and store in 50 mL Falcon tube
In the upcoming steps, this L.B Media will be used
Thawed the cells by keeping the eppendorf containing competent cells at 0 °C on ice
Add 100 ng Plasmid to the Eppendorf
100ng= 0.1 ug
Incubate the Eppendorf for 15 Minutes on Ice
Heat-shock the Eppendorf containing competent cells
42 °C for 45 Seconds
Put Eppendorf on ice for 2-4 Minutes
Add 800 µL of L.B Broth in Eppendorf and incubate at 180 rpm, 37°C, 01:00:00
This step is known as regeneration
1h
After regeneration, harvest the cell by centrifugation 4000 rpm, 37°C, 00:30:00
30m
Discard the supernatant and add 200 µL of L.B Broth , resuspend it till you see a clear solution
(Don't resuspend the cells vigorously, it may damage transformant cells.)
Pour 100 µL of Transformant cells into an agar plate (25 mL plate) and add 10 µL Kanamycin
Incubate it overnight at 180 rpm, 37°C, 24:00:00
1d
Colonies are likely to appear if they have an uptake of plasmid
Induction & Expression Screening (DAY 4-5)
Induction & Expression Screening (DAY 4-5)
1d 20h 15m
1d 20h 15m
Make 40 mL L.B Broth and aliquot 10 mL each in 15 mL Falcon
Pick a healthy, medium-sized, isolated colony from an agar plate and inoculate it in 10 mL of L.B Broth (Primary Culture) ; Incubate at 180 rpm, 37°C, 24:00:00
1d
Take 1% 0.1 µL of the Transformant cells from an overnight culture and inoculate them into 10 mL Falcon (All 4 of them). Incubate them at 180 rpm, 37°C Till it reaches 0.5 OD (Secondary culture)
Add 0.5 mM of IPTG into two Falcon tubes and 0.1 mM of IPTG into two Falcon tubes.
Incubate two Falcon tubes containing 0.1 & 0.5 mM concentration of IPTG at 25 °C 16:00:00
Incubate two Falcon tubes containing 0.1 & 0.5 mM concentration of IPTG at 37 °C 04:00:00
20h
Harvest all 4 Falcon Tubes at 4000 rpm, 37°C, 00:15:00
(At their respective timing)
15m
Run SDS-PAGE and check the protein expression