Evaluation of an E. coli Cell Extract Prepared by Lysozyme‐Assisted Sonication via Gene Expression, Phage Assembly and Proteomics

Abstract

Over the past decades, starting from crude cell extracts, a variety of successful preparation protocols and optimized reaction conditions have been established for the production of cell‐free gene expression systems. One of the crucial steps during the preparation of cell extract‐based expression systems is the cell lysis procedure itself, which largely determines the quality of the active components of the extract. Here we evaluate the utility of an E. coli cell extract, which was prepared using a combination of lysozyme incubation and a gentle sonication step. As quality measure, we demonstrate the cell‐free expression of YFP at concentrations up to 0.6 mg/mL. In addition, we produced and assembled T7 bacteriophages up to a titer of 108 PFU/mL. State‐of‐the‐art quantitative proteomics was used to compare the produced extracts with each other and with a commercial extract. The differences in protein composition were surprisingly small between lysozyme‐assisted sonication (LAS) extracts, but we observed an increase in the release of DNA‐binding proteins for increasing numbers of sonication cycles. Proteins taking part in carbohydrate metabolism, glycolysis, amino acid and nucleotide related pathways were found to be more abundant in the LAS extract, while proteins related to RNA modification and processing, DNA modification and replication, transcription regulation, initiation, termination and the TCA cycle were found enriched in the commercial extract.

Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria

“…With such phage genomes, approximately 0.126 (MS2), 0.05 (CLP-P3), 0.00002 (PhiA1122), 0.0025 (Phi29), and 0.0014 (Goe1) infectious phage particles per genome were obtained. In comparison, the T7 phage can replicate within the cell-free system, resulting in an infectious particle/genome ratio of 2.7 (Vogele et al, 2021).…”

“…It may thus be possible that these type IV genes depend not only on the T7 RNA polymerase but might exploit additional promoter-independent regulatory elements, e.g., RNAs amenable to conformational changes. Besides the identical sequence of protein expression in the cell-free system, the complete DNA replication was also shown, including cleavage of the concatemers at the specific pac sites for the T7 phage (Rustad et al, 2017;Vogele et al, 2021). With respect to producing therapeutic phages, the titers obtained by phactory for PhiA1122, CLB-P3, and MUC-5 targeting Y. pestis, EAEC, and the MDR K. pneumoniae, respectively, were substantially higher (5-fold) than those achieved with amplification in bacterial liquid culture (Figure 4A).…”

“…For the generation of crude cell extract, a protocol described as in Falgenhauer et al (Falgenhauer et al, 2021) was used. In short, the E. coli Rosetta 2 (DE3) cells were cultivated in a 2 l lab-scale bioreactor with pO 2 monitoring and pH control.…”

Complex dynamics in a synchronized cell-free genetic clock

“…Homemade E. coli cell extract was prepared from Rosetta 2(DE3) by sonication based on standard protocols 52 , 53 . The final reaction contained 50 mM Hepes pH 8, 1.5 mM ATP (Roth, #HN35.3) and GTP (Roth, #K056.4), 0.9 mM CTP (Roth, #K057.4) and UTP (Roth, #K055.3), 0.2 mg mL −1 tRNA (Merck, #10109541001), 26 mM coenzyme A (Merck, #C3144-10MG), 0.33 mM NAD + (Merck, #481911), 0.75 mM cAMP (Merck, #A9501-1G), 68 μM folinic acid (Merck, #47612-250MG), 1 mM spermidine (Merck, #S2626-1G), and 30 mM 3-PGA (Merck, #P8877-1G), as an energy source.…”

“…The final reaction contained 50 mM Hepes pH 8, 1.5 mM ATP (Roth, #HN35.3) and GTP (Roth, #K056.4), 0.9 mM CTP (Roth, #K057.4) and UTP (Roth, #K055.3), 0.2 mg mL −1 tRNA (Merck, #10109541001), 26 mM coenzyme A (Merck, #C3144-10MG), 0.33 mM NAD + (Merck, #481911), 0.75 mM cAMP (Merck, #A9501-1G), 68 μM folinic acid (Merck, #47612-250MG), 1 mM spermidine (Merck, #S2626-1G), and 30 mM 3-PGA (Merck, #P8877-1G), as an energy source. The final concentrations of screened components were 53 4 mM Mg-glutamate (Merck, #49605-250G), 60 mM K-glutamate (Merck, #49601-100G), 1.5 mM of each amino acid except leucine (Biozym, #BR1401801), 1.25 mM leucine, 2.5% (w/v) PEG-8000 (Merck, #89510-250G-F), and 0 mM DTT. A final concentration of 2 mM TCEP (Roth, #HN95.1) was added to the buffer solution immediately prior to the experiment to allow storage of buffer reservoirs at ambient temperature 54 .…”