Dec 03, 2025

Public workspacePreparation 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 03, 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)Amount1 L
  • Tryptone Amount10 g (1% of Autoinduction final volume)
  • Yeast ExtractAmount5 g (0.5% of Autoinduction final volume)
  • Ultra-pure water up to Amount930 mL
5052 Solution (50x) Amount500 mL
  • Glycerol Amount125 mL (25%)
  • Glucose Amount12.5 g (2.5%)
  • a-LactoseAmount50 g (10%)
  • Ultra-pure water up to Amount500 mL
Nitrogen-Phosphate Salts (NPS; 20x)Amount1 L
  • Ammonium Sulfate (NH4)2SO4Amount66.07 g Concentration0.5 Molarity (M)
  • Monopotassium phosphate KH2PO4 Amount174.2 g Concentration1 Molarity (M)
  • Disodium phosphate Na2HPO4Amount141.96 g Concentration1 Molarity (M)
  • Ultra-pure water up to Amount1 L
Magnesium Sulphate Amount50 mL Concentration1 Molarity (M)
  • Magnesium Sulphate MgSO4Amount12.32 g Concentration1 Molarity (M)
  • Ultra-pure water up to Amount50 mL
Antibiotics
Amount1 mL of filter-sterilised selected antibiotics at stock concentration.
Troubleshooting
Materials
Expression Lysogeny Broth (LB)Amount1 L
  • Tryptone Amount10 g (1% of Autoinduction final volume)
  • Yeast ExtractAmount5 g (0.5% of Autoinduction final volume)
  • Ultra-pure water up to Amount930 mL
5052 Solution (50x) Amount500 mL
  • Glycerol Amount125 mL (25%)
  • Glucose Amount12.5 g (2.5%)
  • a-LactoseAmount50 g (10%)
  • Ultra-pure water up to Amount500 mL
Nitrogen-Phosphate Salts (NPS; 20x)Amount1 L
  • Ammonium Sulfate (NH4)2SO4Amount66.07 g Concentration0.5 Molarity (M)
  • Monopotassium phosphate KH2PO4 Amount174.2 g Concentration1 Molarity (M)
  • Disodium phosphate Na2HPO4Amount141.96 g Concentration1 Molarity (M)
  • Ultra-pure water up to Amount1 L
Magnesium Sulphate Amount50 mL Concentration1 Molarity (M)
  • Magnesium Sulphate MgSO4Amount12.32 g Concentration1 Molarity (M)
  • Ultra-pure water up to Amount50 mL

Antibiotics
Amount1 mL of filter-sterilised selected antibiotics at stock concentration. Go to for common stock concentrations.

Preparation of Components
1h
Preparation of Expression LB
Add all dry ingredients to a Amount1 L measuring cylinder

Add ultra-pure water up to Amount930 mL

Add magnetic stir bar and mix thoroughly
Add mixuture to Amount1 L Duran bottle, place lid loosely, and autoclave to sterilise with settings: Duration00:20:00 Temperature121 °C

20m
Preparation of 5052 Solution (50x)
Add dry ingredients to a Amount1 L measuring cylinder

Add glycerol to a Amount500 mL measuring cylinder

Add ultra-pure water up to Amount500 mL
Add mixuture to Amount500 mL Duran bottle, place lid loosely, and autoclave to sterilise with settings: Duration00:20:00 Temperature121 °C
20m
Preparation of NPS (20x)
Add dry ingredients to a Amount1 L measuring cylinder

Add ultra-pure water up to Amount1 L
Add magnetic stir bar and mix thoroughly
Add mixuture to Amount1 L Duran bottle, place lid loosely, and autoclave to sterilise with settings: Duration00:20:00 Temperature121 °C
Preparation of Magnesium Sulphate
Add magnesium sulphate to a Amount50 mL measuring cylinder

Add ultra-pure water up to Amount50 mL

Add magnetic stir bar and mix thoroughly
Add mixuture to Amount50 mL Duran bottle, place lid loosely, and autoclave to sterilise with settings: Duration00:20:00 Temperature121 °C
20m
Making Autoinduction LB Broth
After letting all solutions cool to TemperatureRoom temperature :
Add to a Amount5 L baffled flask:
  • Amount1 mL of Concentration1 Molarity (M) Magnesium Sulphate
  • Amount1 mL of appropriate antibiotic
ABC
Stock ConcentrationFinal Concentration
Ampicillin100 mg/mL100 µg/mL
Chloramphenical25 mg/mL25 µg/mL
Kanamycin25 mg/mL25 µg/mL

  • Amount50 mL of 20x NPS
  • Amount20 mL of 50x 5052 Solution
  • 1 bottle of Expression LB (Amount930 mL )

Temperature
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

Optional
Inoculating Autoinduction Broth
Add inoculateAmount1 mL of starter culture to begin expression in desired conditions
Protocol references
Studier, F.W. (2005) Protein Expression and Purification 41:207–234.