Application Note 12

Optimization of BioExpress^® Supplementation of M9 Cultures

Marwa Rhima, Lori C. Neil, and Kevin H. Gardner

Department of Biochemistry, UT Southwestern Medical Center at Dallas, Dallas, TX 75235 USA

Introduction

Uniform labeling of proteins with ¹⁵N and ¹³C has typically been achieved through the use of bacteria grown in minimal media, such as M9, which contain single nitrogen and carbon sources. While this arrangement facilitates the straightforward isotopic replacements of these elements, the growth characteristics of Escherichia coli (E. coli) in these media are somewhat compromised compared to growth in rich media. These effects typically include a drop in maximum cell density, requiring that larger cultures be grown to produce sufficient quantities of protein for NMR study. To avoid the increased expense and time required to prepare, grow, and process such larger cultures, an alternative approach is to supplement minimal media with mixtures of isotopically labeled biomolecules, such as cell lysates. Here we evaluate the effects of adding increasing amounts of one such mixture, BioExpress^® Cell Growth media from Cambridge Isotope Laboratories, Inc., (CIL) to E. coli cultures grown in M9 minimal media. We characterize the beneficial effects of this supplementation on cell growth rates, maximal densities and protein expression, and observe significant benefits in all three of these categories.

Methods

E. coli strain BL21(DE3) (Stratagene) was transformed with the plasmid Gb1-STOP, encoding the streptococcal protein Gβ1 domain (Gb1) under control of a T7 RNA polymerase promoter, and plated on an LB/amp plate. A colony from this plate was inoculated into 5 mL of LB media and allowed to grow for 4 hr at 37°C with vigorous shaking. At this point, the culture was divided into equal parts, centrifuged, and pellets resuspended in M9 media (Table 1) supplemented with various quantities of BioExpress Cell Growth media. For an initial test of growth characteristics without protein induction, these media contained BioExpress at concentrations of 0.5%, 1%, 10%, 20% and a standard M9-only sample. A subsequent test of growth characteristics with protein induction used cultures containing 0.5%, 1%, 2%, 5%, 10% and a standard M9-only sample. For this latter set, protein expression was induced by the addition of 0.5 mM IPTG at a point during the middle of log phase growth (typical A600 values ~0.4-1.2) as determined from the prior test without induction. At all points through these growths, cultures were grown at 37°C with vigorous shaking, and densities were monitored by turbidity at 600 nm (A600). Induced cultures were harvested 3 hr post-induction, at which point they were analyzed for protein expression (SDS-PAGE) and total cell mass (wet weight).

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