Grow and Induce Expression Strains

<aside> <img src="/icons/light-bulb_gray.svg" alt="/icons/light-bulb_gray.svg" width="40px" /> Getting Started

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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).

<aside> <img src="/icons/wrench_gray.svg" alt="/icons/wrench_gray.svg" width="40px" /> Materials and Equipment

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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]

Consumables
Culture tubes	Culture Tube, PS, 14mL, 18x95mm, Sterile, TC Treated, w/ Snap (Vent) Cap	Greiner Bio-One	191160	$258.15	4C to 30C	[link]
50 mL conical tubes	Corning® 50 mL Polypropylene Centrifuge Tubes, Sterile, Racked, CentriStar™ Cap	Corning	430828	$436.88	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]

Equipment
Shaking incubator	New Brunswick Innova 4430 Incubator Shaker	New Brunswick	-	-	4C to 30C	discontinued
Microvolume spectrophotometer	DeNovix DS-11+ Spectrophotometer	DeNovix	DS-11+	unlisted	4C to 30C	[link]
Bench centrifuge	Sorvall X4R Pro-MD, IVD Certified	Sorvall	75009521	$18,270.00	4C to 30C	[link]
-20C Freezer	TSX Series High-Performance -20°C Manual Defrost Freezers	Thermo Scientific	TSX2320FA	unlisted	4C to 30C	[link]
-80C Freezer	TSX Series Ultra-Low Freezers	Thermo Scientific	TSX60086A	unlisted	4C to 30C	[link]

<aside> <img src="/icons/merge_gray.svg" alt="/icons/merge_gray.svg" width="40px" /> Prerequisite Protocols

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[ ] Prepare selective media and inducer: Make Protein Purification Buffers and Media.

<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Protocol

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[ ] Prep overnight cultures.
- [ ] Add 5 mL LB + Kanamycin (50 ug / mL) to 15 mL culture tubes and label.
- [ ] Innoculate your tubes with your expression strain working stock using a pipette tip.
- Notes - you can use glycerol stocks OR fresh colonies as working stocks
- [ ] Incubate cultures overnight at 37C / 225 rpm for between 12 hrs and 16 hrs.
[ ] Perform bulk outgrowth.
- [ ] Back dilute overnight cultures 1:1000 into fresh media (e.g., add 100 uL of overnight and 100 mL LB with Kanamycin to 250 mL Erlenmeyer flasks).
- Notes: leave ≥ 2.5x culture volume in headroom!
- [ ] Incubate back diluted cultures at 37C / 225 rpm / to mid-log phase (OD600 between 0.4 and 0.6 ~ 3.5 hrs).
[ ] Induce protein expression.
- [ ] At mid-log phase, induce your cultures with IPTG to 500 uM (e.g., add 100 uL of IPTG (0.5M) to a 100 mL culture).
- [ ] Incubate induced cultures at 37C / 225 rpm / 4 hr to allow cells to express proteins.
[ ] Centrifuge cells and freeze pellets.
- [ ] While incubating your induced cultures, pre-chill your centrifuge and rotor to 4C.
- [ ] Harvest your cultures by centrifuging at 3200 rcf / 4C / 30 min.
- [ ] Decant supernatant and reserve pellets.
- [ ] Weigh pellets to calculate your biomass yield (gDCM / L).
- [ ] Store pellets at -80C and allow to freeze (at least overnight)
- Notes: take a break!

<aside> <img src="/icons/book_gray.svg" alt="/icons/book_gray.svg" width="40px" /> Resources and References

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Papers
- Original PURE paper
Cell-free translation reconstituted with purified components
- OnePot PURE
A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System

OnePot PURE Cell-Free System | Text Page

<aside> <img src="/icons/megaphone_gray.svg" alt="/icons/megaphone_gray.svg" width="40px" /> Credits

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

Developers