TITLE: Using Skimmed milk as a low-cost alternative to IPTG in plasmid expression induction (LacZ Promoter) 
AUTHORS: Joseph Shenekji

[DESCRIPTION]
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.


[BEFORE_START]
make sure to have skimmed milk without any fat, because it will make it harder to extract and purify your expressed protein.

[GUIDELINES]
i used liquid skimmed milk because it was the only one available, but powdered skimmed milk could work just fine according to references.

[STEPS]
SECTION: process of using Skimmed milk instead of IPTG for induction of protein expression
1. 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.

SECTION: process of using Skimmed milk instead of IPTG for induction of protein expression
2. 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.
SECTION: process of using Skimmed milk instead of IPTG for induction of protein expression
3. Inoculation of Bacteria
Inoculate a single colony of the bacteria containing the lacZ operon into 5 mL of 5 mLsupplemented with the appropriate antibiotic.
Incubate overnight at 37 °C with shaking (-250 rpm).



SECTION: process of using Skimmed milk instead of IPTG for induction of protein expression
7. Harvesting Cells
After the incubation period, centrifuge the culture at 4.000 rpm for 10 min 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.



SECTION: process of using Skimmed milk instead of IPTG for induction of protein expression
4. Subculture
1. The next day, dilute the overnight culture 1:100 into fresh LB broth with antibiotics.
2. Incubate 180 min at 37 °C with shaking until the OD600 reaches 0.4-0.6.

SECTION: process of using Skimmed milk instead of IPTG for induction of protein expression
5. 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. (5 min)
Gently mix the culture to ensure even distribution.

SECTION: process of using Skimmed milk instead of IPTG for induction of protein expression
6. Incubation Post-Induction
Continue incubating the culture at 30-37°C with shaking for an additional 240 min to 360 min, depending on the desired expression level. 

SECTION: process of using Skimmed milk instead of IPTG for induction of protein expression
8. 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.