Rich Medium Composition Affects Escherichia coli Survival, Glycation, and Mutation Frequency during Long-Term Batch Culture

Abstract

Bacteria such as Escherichia coli are frequently grown to high density to produce biomolecules for study in the laboratory. To achieve this, cells can be incubated in extremely rich media that increase overall cell yield. In these various media, bacteria may have different metabolic profiles, leading to changes in the amounts of toxic metabolites produced. We have previously shown that stresses experienced during short-term growth can affect the survival of cells during the long-term stationary phase (LTSP). Here, we incubated cells in LB, 2؋ yeast extract-tryptone (YT), Terrific Broth, or Super Broth medium and monitored survival during the LTSP, as well as other reporters of genetic and physiological change. We observe differential cell yield and survival in all media studied. We propose that differences in long-term survival are the result of changes in the metabolism of components of the media that may lead to increased levels of protein and/or DNA damage. We also show that culture pH and levels of protein glycation, a covalent modification that causes protein damage, affect long-term survival. Further, we measured mutation frequency after overnight incubation and observed a correlation between high mutation frequencies at the end of the log phase and loss of viability after 4 days of LTSP incubation, indicating that mutation frequency is potentially predictive of long-term survival. Since glycation and mutation can be caused by oxidative stress, we measured expression of the oxyR oxidative stress regulator during log-phase growth and found that higher levels of oxyR expression during the log phase are consistent with high mutation frequency and lower cell density during the LTSP. Since these complex rich media are often used when producing large quantities of biomolecules in the laboratory, the observed increase in damage resulting in glycation or mutation may lead to production of a heterogeneous population of plasmids or proteins, which could affect the quality of the end products yielded in some laboratory experiments. E scherichia coli is often used both as a model organism to understand fundamental biological process and as a tool to produce biomolecules, including plasmids and proteins. While many of these applications require relatively short periods of incubation, the E. coli life cycle in batch culture in the laboratory can last for extended periods and include five phases. During the first phase after inoculation into fresh medium, termed the "lag phase," cells are adjusting their metabolism to the new, nutrientrich environment, with little cell division. During the second phase, called the "exponential phase" or "logarithmic phase," cells are replicating at a relatively rapid and constant rate, which can be as fast as 1 cell division every 20 min for many E. coli strains (1). This is followed by the "stationary phase," where cell density ceases to increase, most likely due to some combination of a reduction in available nutrients, a buildup of metabolic wastes or toxins, and/or signals to cease g...

Functional characterization of host toxic EcdB transcription factor protein of echinocandin B biosynthetic gene cluster

“…The full‐length GST‐EcdB protein was expressed in E. coli BL21(DE3) expression host by optimizing different expression conditions. A group of investigators revealed that protein expression or toxic protein expression can be improved by a subsequent alteration of IPTG concentration , temperature , using rich medium , induction at higher cell density , and induction on lawn cells in place of seed culture . We first tested IPTG (1 µM, 10 µM, 0.5 mM, and 1 mM) and lawn cells (10 µM IPTG)‐dependent induction at 37 °C.…”

“…The uninduced construct was treated as a negative control in the entire study. Based on previous reports, the full‐length (GST:EcdB) protein was expressed by optimizing three different parameters such as IPTG concentration, temperature, and growth medium . In spite of that, IPTG induction at lawn cells and higher cell density (1 OD) was also tested as investigated previously .…”

“…For optimizing IPTG concentration, cells were induced with 1 µM, 10 µM, 0.5 mM, 1 mM IPTG at 0.5–0.6 OD for 3 H. In addition, protein expression was also performed at different incubation temperature such as 37, 30, 16, 10, and 4 °C for 3, 4, 24, 48, and 72 H, respectively . Different rich medium such as Super Broth (SB) (35 g tryptone, 20 g yeast extract, 5 g NaCl), Super Optimal Broth (SOB) (20 g bacto tryptone, 5 g yeast extract, 0.5 g NaCl, 0.186 g KCl, 0.952 g MgCl 2 , 2.467 g MgSO 4 ), 2x‐YT (16 g bacto tryptone, 10 g yeast extract, 5 g NaCl), Terrific Broth (TB) (HiMedia), and LB (G‐Biosciences) were also used for protein expression . After the completion of induction experiment at different tested conditions, cells were harvested by centrifugation at 10,000 rpm for 5 Min.…”

Escherichia colihas a Unique Transcriptional Program in Long-Term Stationary Phase

“…This allows the remaining 67 ~1% of cells to enter long-term stationary phase (LTSP), where they can use the 68 detritus of lysed cells as carbon and energy sources (10-12). The stresses of long-69 term stationary phase likely include high pH, high oxidative stress but low oxygen, 70 and a lack of readily metabolized nutrients, however none of these are 71 characterized completely (10, 13,14). We have previously shown that cells with 72 mutations that may help cope with these stresses are selected for during this 73 phase, and that as populations continue further into LTSP, there is turnover of 74 different mutant genotypes depending on the medium conditions at any given time 75 (7,9).…”

The Effects of Temperature and Culture Medium Composition on Protein Recombinant JSU-pET Expression

“…The expression of recombinant protein was influenced by severalfactors, IPTG concentration, Cell Optical Density, culture media composition (Studier 2005;Uhoraningonga et al, 2018), and temperature (Song et al, 2012).The low temperature cultivation (<28 o C) after induction with IPTG will formed protein in a soluble form and increase the yield of protein (Singh et al, 2015;Larentis et al, 2014;Carrio et al, 2005) While media composition will give high density cell culture or the induce the death of E. coli cells (Farrel and Finkel 2003). According to Kram and Finkel (2015), The effect of media compositionswill lead the oxidative stress, increasing of pH, the death of cells during log phase and changes in long-term survival patterns…”