Sep 07, 2024
Using Skimmed milk as a low-cost alternative to IPTG in plasmid expression induction (LacZ Promoter)
- 1department of biotechnology engineering, faculty of technical engineering, university of Aleppo
- Reclone.org (The Reagent Collaboration Network)Tech. support email: protocols@recode.org
Protocol Citation: Joseph Shenekji 2024. Using Skimmed milk as a low-cost alternative to IPTG in plasmid expression induction (LacZ Promoter) . protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk81z6l5r/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: Working
We use this protocol and it's working
Created: September 07, 2024
Last Modified: September 07, 2024
Protocol Integer ID: 107101
Keywords: skimmed milk, alternatives, low-cost, IPTG, expression, protein
Disclaimer
the protocol is only a proof of concept, it needs to be enhanced and measured accurately.
we do not have a spectrophotometer or turbidity meter, so everything is evaluated visually as a primary method.
Abstract
This protocol provides a framework for using skimmed milk as an alternative to IPTG for inducing lacZ operon expression in bacteria. Adjustments may be necessary based on specific experimental conditions or bacterial strains used.
Image Attribution
image is taken by joseph shenekji
Guidelines
i used liquid skimmed milk because it was the only one available, but powdered skimmed milk could work just fine according to references.
Materials
- Bacterial strain containing the lacZ operon (e.g., E. coli)
- LB (Luria-Bertani) broth
- Skimmed milk powder or liquid
- Antibiotics (e.g., ampicillin or kanamycin, as appropriate)
- Incubator
- Shaking incubator
- Centrifuge
- Culture flasks
- Sterile water
Safety warnings
do not touch the skimmed milk after evaporating water in the microwave, it will be extremely hot.
Ethics statement
this method uses skimmed milk provided from supermarkets.
Before start
make sure to have skimmed milk without any fat, because it will make it harder to extract and purify your expressed protein.
process of using Skimmed milk instead of IPTG for induction of protein expression
process of using Skimmed milk instead of IPTG for induction of protein expression
1m
1m
read the label for the skimmed milk
it should contain a percentage of carbohydrates (sugars) it is usually 5% for each 100 ml. if you have powdered skimmed milk things are easier, where you can use 1.0% (w/v) of skimmed milk for the induction.
1m
Preparation of Skimmed Milk Solution
- Dissolve skimmed milk powder in sterile water to create a 5% (w/v) solution.
- Sterilize the solution by autoclaving or filter sterilization.
Inoculation of Bacteria
- Inoculate a single colony of the bacteria containing the lacZ operon into 5 mL of 5 mL supplemented with the appropriate antibiotic.
- Incubate overnight at 37 °C with shaking (-250 rpm ).
Subculture
1. The next day, dilute the overnight culture 1:100 into fresh LB broth with antibiotics.
2. Incubate 03:00:00 at 37 °C with shaking until the OD600 reaches 0.4-0.6.
3h
Induction with Skimmed Milk
- Once the culture reaches the desired OD600, add skimmed milk solution to a final concentration of 1% (w/v) if it is powdered, in case it is liquid, put 20 ml of skimmed milk in the microwave and evaporate the water until it gives a sludge of ~1ml, wait for it to cool down then add it to the culture it will give approximately 1% w/v of lactose. (00:05:00 )
- Gently mix the culture to ensure even distribution.
5m
Incubation Post-Induction
Continue incubating the culture at 30-37°C with shaking for an additional 04:00:00 to 06:00:00 , depending on the desired expression level.
10h
Harvesting Cells
- After the incubation period, centrifuge the culture at 4.000 rpm for 00:10:00 at4 °C to pellet the cells.
- Discard the supernatant and resuspend the cell pellet in an appropriate buffer (e.g., PBS or lysis buffer) for further analysis or protein extraction.
10m
final result looked like this
we used IPTG 0.1 M and skimmed milk 1% W/V to induce Lac Z operon to express GFP, and results came according to the image.
comparing results of using IPTG in inducing GFP expression in LacZ plasmid, and skimmed milk culture of the same bacteria and expression. skimmed milk shows better glow visually under uv.
Protocol references
Bashir, H., Ahmed, N., Khan, M.A., Zafar, A.U., Tahir, S., Khan, M.I., Khan, F. and Husnain, T. (2016), Simple procedure applying lactose induction and one-step purification for high-yield production of rhCIFN. Biotechnology and Applied Biochemistry, 63: 708-714. https://doi.org/10.1002/bab.1426
Khani, Mohammad-Hosein, and Masoumeh Bagheri. "Skimmed milk as an alternative for IPTG in induction of recombinant protein expression." Protein expression and purification 170 (2020): 105593.
Tian, H., Tang, L., Wang, Y. et al. Lactose Induction Increases Production of Recombinant Keratinocyte Growth Factor-2 in Escherichia coli . Int J Pept Res Ther 17, 123–129 (2011). https://doi.org/10.1007/s10989-011-9249-9