Editing S30 E. coli extract (section)

== Protocol 2 ==

https://www.nature.com/articles/srep08663.pdf

'''Materials.''' E. coli BL21 StarTM (DE3) strain was purchased from Life Technologies (Grand Island, NY). Strain C495 (also reported as MCJ.495) is a genomically engineered variant of K12 MG1655 described previously48. E. coli total tRNA mixture (from strain MRE600) was purchased from Roche Applied Science (Indianapolis, IN). ATP, GTP, CTP, UTP, Phosphoenolpyruvate, 20 amino acids and other materials were purchased from Sigma (St. Louis, MO) without further purification. sfGFP was cloned into pY71 vector using NdeI and SalI restriction site and used as model protein in this study. T7 RNA Polymerase was obtained by affinity tag purification56.

'''Preparation of cell extracts'''<br/>E. coli BL21 Star™ (DE3) and C495 cells were grown in 2 × YTPG media at 34°C. We grew the strains at 34°C such that we could compare the strains. The MG1655-based C495 strain harbors a temperature sensitive λ-Red recombinase that requires growth at a lower temperature. Antibiotics were not used during cell growth. The cells were grown in a BIOSTAT C-plus fermentor (Sartorious AG, Goettingen, Germany) at 600 rpm for 10 L large-scale cultures. For preparing 1 L scale cultures, cells were grown in 2.5 L baffled tunair shake flasks (IBI Scientific, Peosta, IA) in a 34°C incubator with vigorous shaking at 250 rpm. For smaller scale cultivation (50 to 100 mL culture), 300 mL baffled tunair flasks were used. Unless otherwise specified in the text, the cultured cells were monitored by spectrophotometry (Genesys 10S UV-Vis, Thermo Fisher Scientific, Waltham, MA) until OD600 reached at 3.0. The maximum OD600 for BL21 Star™ (DE3) and C495 strains measured after 22 h culture that reached saturation were ~10 and ~8, respectively. The cells were harvested in the middle of exponential growth phase by centrifuging at 5000 RCF at 4°C for 15 min and were washed three times with cold Buffer A. Buffer A contained 10 mM Tris-acetate (pH 8.2), 14 mM magnesium acetate, 60 mM potassium glutamate, and 2 mM dithiothreitol. After final wash and centrifugation, the pelleted wet cells were weighed, flash frozen in liquid nitrogen, and stored at −80°C. The thawed cells were suspended in 1 mL of Buffer A per 1 g of wet cell mass. For obtaining standard “control” large-scale cultured cell extract, suspended cells were disrupted by EmulsiFlex-C3 homogenizer (Avestin, Ottawa, Canada) with single pass at a variable pressure of 20,000 to 25,000 psig. In order to remove cell debris and insoluble components in the cell lysate, the lysate was centrifuged twice at 30,000 RCF at 4°C for 30 min. The supernatant was then incubated at 37°C for 60 min with gentle shaking (250 rpm) and centrifuged at 15,000 RCF at 4°C for 15 min. In order to lyse cells by sonication, thawed and suspended cells were transferred into 1.5 mL microtube and placed in an ice-water bath to minimize heat damage during sonication. The cells were lysed using a Q125 Sonicator (Qsonica, Newtown, CT) with 3.175 mm diameter probe at frequency of 20 kHz and 50% of amplitude. The input energy (Joules) was monitored and recorded during sonication. The lysate was then centrifuged once at 12,000 RCF at 4°C for 10 min. The run-off reaction (37°C at 250 rpm) and second centrifugation (10,000 RCF at 4°C for 10 min) were performed for strain C495. However, in the case of strain BL21 Star™ (DE3), the run-off reaction and second centrifugation step were not needed. The total amount of protein in cell extract was quantified by Bradford assay. All extracts contained 40.7 ± 2.6 mg/mL of total E. coli proteins. All of prepared cell extract was flash frozen in liquid nitrogen and stored at −80°C until use.

'''Cell-free protein synthesis'''<br/>The CFPS reactions were carried out in a 1.5 mL microtube in the incubator. The standard reaction mixture for CFPS consists of the following components in a final volume of 15 μL: 1.2 mM ATP; 0.85 mM each of GTP, UTP, and CTP; 34.0 μg mL−1 L-5-formyl-5, 6, 7, 8-tetrahydrofolic acid (folinic acid); 170.0 μg mL−1 of E. coli tRNA mixture; 130 mM potassium glutamate; 10 mM ammonium glutamate; 12 mM magnesium glutamate; 2 mM each of 20 amino acids; 10 μM of L-[14C(U)]-leucine (11.1 GBq mmol−1, PerkinElmer, Waltham, MA); 0.33 mM nicotinamide adenine dinucleotide (NAD); 0.27 mM coenzyme-A (CoA); 1.5 mM spermidine; 1 mM putrescine; 4 mM sodium oxalate; 33 mM phosphoenolpyruvate (PEP); 13.3 μg mL−1 plasmid; 100 μg mL−1 T7 RNA polymerase, and 27% v/v of cell extract. The CFPS reactions were carried out at 37°C for 4 hours.