Dec 02, 2025

Preparation of Autoinduction Lysogeny broth (LB) for Protein Expression

DOI
https://dx.doi.org/10.17504/protocols.io.eq2ly46ymlx9/v1
  • Alexander Tynan1,
  • Jeff Nanson1
  • 1Rural Health Research Institute/Charles Sturt University
Alexander Tynan
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DOI: https://dx.doi.org/10.17504/protocols.io.eq2ly46ymlx9/v1
Protocol Citation: Alexander Tynan, Jeff Nanson 2025. Preparation of Autoinduction Lysogeny broth (LB) for Protein Expression. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly46ymlx9/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 03, 2025
Last Modified: December 02, 2025
Protocol  Integer ID: 226295
Keywords: Autoinduction, media, LB, protein expression, preparation of autoinduction lysogeny broth, t7 rna polymerase while glycerol, autoinduction lysogeny broth, protein expression auto, t7 rna polymerase, recombinant protein expression, lactose, sterile concentrate, preparation, glucose, streamlined recipe
Abstract
Auto-induction LB (ZYM-5052) enables high-level recombinant protein expression in E. coli without manual IPTG induction. This protocol provides a streamlined recipe and culture workflow adapted from Studier (2005) for T7-based systems (e.g., pET vectors in BL21(DE3) and derivatives). Medium is assembled from sterile concentrates: 20× NPS salts, 50× “5052” carbon mix (glycerol, glucose, lactose), 1 mM MgSO₄, and appropriate antibiotic(s). Media used in baffled flasks at ≤20–25% fill to ensure aeration. Glucose supports initial growth and represses expression; as it is exhausted, lactose automatically induces T7 RNA polymerase while glycerol sustains continued growth.
Materials
Expression Lysogeny Broth (LB)1 L
  • Tryptone 10 g (1% of Autoinduction final volume)
  • Yeast Extract5 g (0.5% of Autoinduction final volume)
  • Ultra-pure water up to 930 mL
5052 Solution (50x) 500 mL
  • Glycerol 125 mL (25%)
  • Glucose 12.5 g (2.5%)
  • a-Lactose50 g (10%)
  • Ultra-pure water up to 500 mL
Nitrogen-Phosphate Salts (NPS; 20x)1 L
  • Ammonium Sulfate (NH4)2SO466.07 g 0.5 Molarity (M)
  • Monopotassium phosphate KH2PO4 174.2 g 1 Molarity (M)
  • Disodium phosphate Na2HPO4141.96 g 1 Molarity (M)
  • Ultra-pure water up to 1 L
Magnesium Sulphate 50 mL 1 Molarity (M)
  • Magnesium Sulphate MgSO412.32 g 1 Molarity (M)
  • Ultra-pure water up to 50 mL
Antibiotics
1 mL of filter-sterilised selected antibiotics at stock concentration.
Materials
Expression Lysogeny Broth (LB)1 L
  • Tryptone 10 g (1% of Autoinduction final volume)
  • Yeast Extract5 g (0.5% of Autoinduction final volume)
  • Ultra-pure water up to 930 mL
5052 Solution (50x) 500 mL
  • Glycerol 125 mL (25%)
  • Glucose 12.5 g (2.5%)
  • a-Lactose50 g (10%)
  • Ultra-pure water up to 500 mL
Nitrogen-Phosphate Salts (NPS; 20x)1 L
  • Ammonium Sulfate (NH4)2SO466.07 g 0.5 Molarity (M)
  • Monopotassium phosphate KH2PO4 174.2 g 1 Molarity (M)
  • Disodium phosphate Na2HPO4141.96 g 1 Molarity (M)
  • Ultra-pure water up to 1 L
Magnesium Sulphate 50 mL 1 Molarity (M)
  • Magnesium Sulphate MgSO412.32 g 1 Molarity (M)
  • Ultra-pure water up to 50 mL

Antibiotics
1 mL of filter-sterilised selected antibiotics at stock concentration. for common stock concentrations.

Preparation of Components
1h
Preparation of Expression LB
Add all dry ingredients to a 1 L measuring cylinder

Add ultra-pure water up to 930 mL

Add magnetic stir bar and mix thoroughly
Add mixuture to 1 L Duran bottle, place lid loosely, and autoclave to sterilise with settings: 00:20:00 121 °C

20m
Preparation of 5052 Solution (50x)
Add dry ingredients to a 1 L measuring cylinder

Add glycerol to a 500 mL measuring cylinder

Add ultra-pure water up to 500 mL
Add mixuture to 500 mL Duran bottle, place lid loosely, and autoclave to sterilise with settings: 00:20:00 121 °C
20m
Preparation of NPS (20x)
Add dry ingredients to a 1 L measuring cylinder

Add ultra-pure water up to 1 L
Add magnetic stir bar and mix thoroughly
Add mixuture to 1 L Duran bottle, place lid loosely, and autoclave to sterilise with settings: 00:20:00 121 °C
Preparation of Magnesium Sulphate
Add magnesium sulphate to a 50 mL measuring cylinder

Add ultra-pure water up to 50 mL

Add magnetic stir bar and mix thoroughly
Add mixuture to 50 mL Duran bottle, place lid loosely, and autoclave to sterilise with settings: 00:20:00 121 °C
20m
Making Autoinduction LB Broth
After letting all solutions cool to Room temperature :
Add to a 5 L baffled flask:
  • 1 mL of 1 Molarity (M) Magnesium Sulphate
  • 1 mL of appropriate antibiotic
ABC
Stock ConcentrationFinal Concentration
Ampicillin100 mg/mL100 µg/mL
Chloramphenical25 mg/mL25 µg/mL
Kanamycin25 mg/mL25 µg/mL

  • 50 mL of 20x NPS
  • 20 mL of 50x 5052 Solution
  • 1 bottle of Expression LB (930 mL )

Alternatively, measure out appropriate volumes of autoinduction media into other sizes of baffled flasks:

AB
Media Volume (mL)Flask Size (mL)
125500
2501000
5002000
Media to flask ratio generally 1:4

Inoculating Autoinduction Broth
Add inoculate1 mL of starter culture to begin expression in desired conditions
Protocol references
Studier, F.W. (2005) Protein Expression and Purification 41:207–234.