Grow and Induce Expression Strains

Getting Started

You want to purify proteins. First, you’re going to have to make some. We make proteins using bacterial strains that carry our protein of interest on an expression plasmid (here: pET28a). These expression plasmids put a gene of interest under the transcription of an inducible promoter (e.g., pT7). This allows us to first (1) grow our bacteria quickly to a high density, without the metabolic load of making a lot of proteins, then to (2) induce the overexpression of our protein of interest. Making so much protein is toxic to the cells, so we only want to induce expression once our culture is grown out (OD600 ~ 0.5).

Materials and Equipment

Name	Product	Manufacturer	Part #	Price	Storage Conditions	Link
Media
LB	Luria Broth (Miller's LB Broth), Non-Sterile, 6.8 - 7.2, Molecular Biology Grade, suitable for regular E.coli culture, Powder	Sigma-Aldrich	L3522-1KG	$221	4C to 30C	[link]
IPTG	Isopropyl β-D-thiogalactoside (IPTG), Powder, ≥99% (TLC), ≤0.1% Dioxane	Sigma-Aldrich	I6758-1G	$89.90	-25C to -15C	[link]
Kanamycin	BioReagent Kanamycin sulfate, ≥750 ug/mg, From Streptomyces kanamyceticus, Suitable for cell culture, Suitable for plant cell culture, Powder	Sigma-Aldrich	K1377-1G	$47.70	4C to 30C	[link]

Culture Tubes
Culture tubes	Culture Tube, PS, 14mL, 18x95mm, Sterile, TC Treated, w/ Snap (Vent) Cap	Greiner Bio-One	191160	$258.15	4C to 30C	[link]

Flasks
250 mL baffled flasks	PYREX® 250 mL Delong Shaker Erlenmeyer Flask with Baffles	Pyrex	4444-250	$188.26	4C to 30C	[link]
flask closures	Chemglass Life Sciences Closure, 38mm, Stainless Steel	Chemglass Life Sciences	Chemglass Life Sciences Closure, 38mm, Stainless Steel	$100.75	4C to 30C	[link]

Prerequisite Protocols

Prepare selective media and inducer:

Make Protein Purification Buffers and Media.

Protocol

Prep overnight cultures.

Add 5 mL LB + kanamycin (50 ug / mL) to 15 mL culture tubes. Label each tube.

Poke your expression strain working stock with a pipette tip.

Eject the whole tip into each culture tube.

Incubate cultures overnight at 37C / 250 rpm for between 10 hrs and 16 hrs.

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Notes - you can work from glycerol stocks OR fresh colonies.

Perform bulk outgrowth.

Back dilute overnights 1:1000 into fresh media (e.g., add 100 uL of overnight and 100 mL LB with Kanamycin to 250 mL Erlenmeyer flasks).

Incubate back diluted cultures at 37C / 250 rpm / to mid-log phase (OD600 between 0.4 and 0.6 ~ 3.5 hrs.)

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Notes: leave ≥ 2.5x culture volume in headroom!

Induce protein expression.

Once bacterial cultures reach midlog phase (OD600 between 0.4 and 0.6) add IPTG to 500 uM. To do this, we added IPTG from 0.5M aliquots (1000x) at 1:1000. E.g., for 100 mL of culture, we add 100 uL of IPTG at 0.5M.

Incubate induced cultures at 37C / 250 rpm / 4 hr to allow cells to express proteins.

Centrifuge cells and freeze pellets.

While induced cultures are incubating, pre-chill centrifuge and rotor to 4C

Pour 50 mL of each culture into 50 mL centrifuge tubes and label each (2x50 mL tubes per 100 mL culture).

Centrifuge cultures at 3200 rcf / 4C / 30 min.

Decant supernatant and reserve pellets.

Store pellets at -80C and allow to freeze (at least overnight)

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Notes: freezing pellets may help lyse cells.

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Notes: take a break!

Resources and References

Papers

Original PURE paper

Cell-free translation reconstituted with purified components

Nature Biotechnology - Cell-free translation reconstituted with purified components

doi.org

Cell-free translation reconstituted with purified components

OnePot PURE

A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System

We demonstrate a simple, robust, and low-cost method for producing the PURE cell-free transcription–translation system. Our OnePot PURE system achieved a protein synthesis yield of 156 μg/mL at a cost of 0.09 USD/μL, leading to a 14-fold improvement in cost normalized protein synthesis yield over existing PURE systems. The one-pot method makes the PURE system easy to generate and allows it to be readily optimized and modified.

doi.org

A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System

OnePot PURE Cell-Free System | Text Page

Scientific Article | The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for...

dx.doi.org

OnePot PURE Cell-Free System | Text Page

Credits

Yan Zhang, Zoila Jurado, and Miki Yun (Richard Murray Lab, Caltech)

Developers