Process optimization for scalable E. coli extract preparation for cell-free protein synthesis

Dopp, Jared L.; Reuel, Nigel F.

doi:10.1016/j.bej.2018.06.021

Cited by 35 publications

(80 citation statements)

References 25 publications

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“…The time required to express protein from this paper's in vitro technique is easily kept within 24 hr, if one has already grown and processed the cell extract or obtained this from a commercial vendor. Our previous work enables scalable, lyophilized extract preparation that performs as well as smaller‐scale, tip‐sonicated methods and outperforms commercial extract (Dopp & Reuel, ). Moreover, it can be grown in > 100 g (wet cell mass) batches that are conveniently lyophilized, stored, and easily sourced to requesting partners.…”

Section: Resultsmentioning

confidence: 99%

“…The cost of protein prototyping from this in vitro method is competitive especially when one uses scalable extract preparation techniques as noted above (Dopp & Reuel, ) and minimal reagent recipes (Cai et al, ; Calhoun & Swartz, ; Jewett & Swartz, ; Kim et al, ). When analyzing the cost per gram of sfGFP produced (Supporting Information Figures S10), we found a minimalist reagent mix, like those used in previous works (Cai et al, ; Dopp & Reuel, ), produced sfGFP at 110 µg/ml (found by fluorescence calibration curve Figure S12) after 4 hr of expression which resulted in $61/mg sfGFP. When using a vendor kit, such as Promega T7 S30 High‐Yield with the minimalist, circular templates a 64% decrease in protein yield (40 µg/ml) was observed (Supporting Information Figure S11) which resulted in a 135‐fold increase in cost ($8,265/mg sfGFP; Supporting Information Figure S13).…”

Section: Resultsmentioning

confidence: 99%

“…All cell‐free reactions were conducted at 37°C in volumes of 15 µl using the following: 30% v/v E. coli extract (produced according to our previously published work on scalable extract [Dopp and Reuel, ]), 10 ng/µl of RCA DNA, 6 µl T7 S30 High‐Yield Premix (Promega), and double distilled water. Lyophilized extract was reconstituted with double distilled water according to previous experiments H 2 O (ml) = (mass of lyophilized extract [g])(1.5 ml/0.094 g) (Dopp & Reuel, ).…”

Section: Methodsmentioning

confidence: 99%

“…The resulting, viscous mixture is diluted by adding 14 µl ddH 2 O per tube (total volume 25 µl) and all are then combined, and purified with the Zymo kit, eluted with 50 µl of water with a typical final RCA template concentration of 70 ng/µl. All cell-free reactions were conducted at 37°C in volumes of 15 µl using the following: 30% v/v E. coli extract (produced according to our previously published work on scalable extract [Dopp and Reuel, 2018]), 10 ng/µl of RCA DNA, 6 µl T7 S30 High-Yield Premix (Promega), and double distilled water. Lyophilized extract was reconstituted with double distilled water according to previous experiments H 2 O (ml) = (mass of lyophilized extract [g])(1.5 ml/ 0.094 g) (Dopp & Reuel, 2018).…”

Section: Methodsmentioning

confidence: 99%

“…Our previous work enables scalable, lyophilized extract preparation that performs as well as smaller-scale, tip-sonicated methods and outperforms commercial extract (Dopp & Reuel, 2018). Moreover, it can be grown in > 100 g (wet cell mass) batches that are conveniently lyophilized, stored, and easily sourced to requesting partners.…”

Section: Advantages and Limitations: Time Cost And Modularitymentioning

confidence: 99%

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Rapid prototyping of proteins: Mail order gene fragments to assayable proteins within 24 hours

Dopp

Rothstein

Mansell

et al. 2019

Biotech & Bioengineering

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In this study, we present a minimal template design and accompanying methods to produce assayable quantities of custom sequence proteins within 24 hr from receipt of inexpensive gene fragments from a DNA synthesis vendor. This is done without the conventional steps of plasmid cloning or cell‐based amplification and expression. Instead the linear template is PCR amplified, circularized, and isothermally amplified using a rolling circle polymerase. The resulting template can be used directly with cost‐optimized, scalably‐manufactured Escherichia coli extract and minimal supplement reagents to perform cell‐free protein synthesis (CFPS) of the template protein. We demonstrate the utility of this template design and 24 hr process with seven fluorescent proteins (sfGFP, mVenus, mCherry, and four GFP variants), three enzymes (chloramphenicol acetyltransferase, a chitinase catalytic domain, and native subtilisin), a capture protein (anti‐GFP nanobody), and 2 antimicrobial peptides (BP100 and CA(1–7)M(2–9)). We detected each of these directly from the CFPS reaction using colorimetric, fluorogenic, and growth assays. Of especial note, the GFP variant sequences were found from genomic screening data and had not been expressed or characterized before, thus demonstrating the utility of this approach for phenotype characterization of sequenced libraries. We also demonstrate that the rolling circle amplified version of the linear template exhibits expression similar to that of a complete plasmid when expressing sfGFP in the CFPS reaction. We evaluate the cost of this approach to be $61/mg sfGFP for a 4 hr reaction. We also detail limitations of this approach and strategies to overcome these, namely proteins with posttranslational modifications.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Advantages and Limitations: Time Cost And Modularitymentioning

confidence: 99%

See 3 more Smart Citations

Rapid prototyping of proteins: Mail order gene fragments to assayable proteins within 24 hours

Dopp

Rothstein

Mansell

et al. 2019

Biotech & Bioengineering

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Optimization of E. coli tip‐sonication for high‐yield cell‐free extract using finite element modeling

2021

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Optimal tip sonication settings, namely tip position, input power, and pulse durations, are necessary for temperature sensitive procedures like preparation of viable cell extract. In this paper, the optimum tip immersion depth (20–30% height below the liquid surface) is estimated which ensures maximum mixing thereby enhancing thermal dissipation of local cavitation hotspots. A finite element (FE) heat transfer model is presented, validated experimentally with (R2 > 97%) and used to observe the effect of temperature rise on cell extract performance of Escherichia coli BL21 DE3 star strain and estimate the temperature threshold. Relative yields in the top 10% are observed for solution temperatures maintained below 32°C; this reduces below 50% relative yield at temperatures above 47°C. A generalized workflow for direct simulation using the CONSOL code as well as master plots for estimation of sonication parameters (power input and pulse settings) is also presented.

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Rapid RNase inhibitor production to enable low‐cost, on‐demand cell‐free protein synthesis biosensor use in human body fluids

Soltani

Hunt

Bundy

2021

Biotech & Bioengineering

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Human body fluids contain biomarkers which are used extensively for prognostication, diagnosis, monitoring, and evaluation of different treatments for a variety of diseases and disorders. The application of biosensors based on cell‐free protein synthesis (CFPS) offers numerous advantages including on‐demand and at‐home use for fast, accurate detection of a variety of biomarkers in human fluids at an affordable price. However, current CFPS‐based biosensors use commercial RNase inhibitors to inhibit different RNases present in human fluids and this reagent is approximately 90% of the expense of these biosensors. Here the flexible nature of Escherichia coli‐lysate‐based CFPS was used for the first time to produce murine RNase Inhibitor (m‐RI) and to optimize its soluble and active production by tuning reaction temperature, reaction time, reduced potential, and addition of GroEL/ES folding chaperons. Furthermore, RNase inhibition activity of m‐RI with the highest activity and stability was determined against increasing amounts of three human fluids of serum, saliva, and urine (0%–100% v/v) in lyophilized CFPS reactions. To further demonstrate the utility of the CFPS‐produced m‐RI, a lyophilized saliva‐based glutamine biosensor was demonstrated to effectively work with saliva samples. Overall, the use of CFPS‐produced m‐RI reduces the total reagent costs of CFPS‐based biosensors used in human body fluids approximately 90%.

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Process optimization for scalable E. coli extract preparation for cell-free protein synthesis

Cited by 35 publications

References 25 publications

Rapid prototyping of proteins: Mail order gene fragments to assayable proteins within 24 hours

Rapid prototyping of proteins: Mail order gene fragments to assayable proteins within 24 hours

Optimization of E. coli tip‐sonication for high‐yield cell‐free extract using finite element modeling

Rapid RNase inhibitor production to enable low‐cost, on‐demand cell‐free protein synthesis biosensor use in human body fluids

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