&lt;em&gt;Escherichia coli&lt;/em&gt;-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Levine, Max Z.; Gregorio, Nicole E.; Jewett, Michael C.; Watts, Katharine R.; Oza, Javin P.

doi:10.3791/58882-v

Cited by 16 publications

(22 citation statements)

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“…The culture was decanted into 1 L bottles and cells were pelleted by centrifugation at 5,000 X g for 15 minutes at 4 °C. The cell pellets were washed once with 25 mL S30A buffer (14 mM Mg-glutamate, 60 mM K-glutamate, 50 mM Tris) and re-centrifuged for 10 minutes at 7,000 X g at 4 °C, following a report that one wash is sufficient to maintain good expression activity in the final extract [74]. The pellets were resuspended in S30A buffer at a ratio of 1 mL buffer/g pellet, transferred to 1.7 mL Eppendorf tubes, and sonicated on ice at 50% amplitude for 1 minute in six 10-second pulses using a QSonica Q125 small-tip probe.…”

Section: Methodsmentioning

confidence: 99%

Section: Cell Extract Preparationmentioning

confidence: 99%

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At-home, cell-free synthetic biology education modules for transcriptional regulation and environmental water quality monitoring

Jung

Rasor

Rybnicky

et al. 2023

Preprint

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As the field of synthetic biology expands, the need to grow and train science, technology, engineering, and math (STEM) practitioners is essential. However, the lack of access to hands-on demonstrations has led to inequalities of opportunity and practice. In addition, there is a gap in providing content that enables students to make their own bioengineered systems. To address these challenges, we develop four shelf-stable cell-free biosensing educational modules that work by just-adding-water and DNA to freeze-dried crude extracts of Escherichia coli. We introduce activities and supporting curricula to teach the structure and function of the lac operon, dose-responsive behavior, considerations for biosensor outputs, and a 'build-your-own' activity for monitoring environmental contaminants in water. We piloted these modules with K-12 teachers and 130 high school students in their classrooms (and at home) without professional laboratory equipment or researcher oversight. This work promises to catalyze access to interactive synthetic biology education opportunities.

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Section: Methodsmentioning

confidence: 99%

Section: Cell Extract Preparationmentioning

confidence: 99%

At-home, cell-free synthetic biology education modules for transcriptional regulation and environmental water quality monitoring

Jung

Rasor

Rybnicky

et al. 2023

Preprint

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“…Cell extracts were prepared using previously described methods with modifications (Karim & Jewett, 2016; Levine et al, 2019). E. coli Rosetta(DE3) cells were grown in 2 × YTPG media (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, 7 g/L potassium phosphate monobasic, 3 g/L potassium phosphate dibasic, 18 g/L glucose).…”

Section: Methodsmentioning

confidence: 99%

“…All CFPS reactions used a modified PANOx‐SP formula described in previous pubications with modifications (Jewett & Swartz, 2004; Levine et al, 2019). A 15 μl CFPS reaction in a 1.5 ml microcentrifuge tube was prepared by mixing the following components: ATP (1.8 mM); GTP, UTP, and CTP (1.3 mM each); folinic acid (0.1 mM); oxalic acid (4 mM), E. coli tRNA mixture (260 μg/ml); 20 standard amino acids (2 mM each); NAD (0.4 mM); coenzyme A (0.27 mM); phosphoenolpyruvate (PEP; 33 mM); spermidine (1.5 mM); putrescine (1 mM); potassium glutamate (130 mM); magnesium glutamate (10 mM); HEPES (57 mM), and cell extract (10 μl).…”

Section: Methodsmentioning

confidence: 99%

Rapid prototyping enzyme homologs to improve titer of nicotinamide mononucleotide using a strategy combining cell‐free protein synthesis with split GFP

Yuan

Liao

et al. 2023

Biotech & Bioengineering

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Engineering biological systems to test new pathway variants containing different enzyme homologs is laborious and time-consuming. To tackle this challenge, a strategy was developed for rapidly prototyping enzyme homologs by combining cellfree protein synthesis (CFPS) with split green fluorescent protein (GFP). This strategy featured two main advantages: (1) dozens of enzyme homologs were parallelly produced by CFPS within hours, and (2) the expression level and activity of each homolog was determined simultaneously by using the split GFP assay. As a model, this strategy was applied to optimize a 3-step pathway for nicotinamide mononucleotide (NMN) synthesis. Ten enzyme homologs from different organisms were selected for each step. Here, the most productive homolog of each step was identified within 24 h rather than weeks or months. Finally, the titer of NMN was increased to 1213 mg/L by improving physiochemical conditions, tuning enzyme ratios and cofactor concentrations, and decreasing the feedback inhibition, which was a more than 12-fold improvement over the initial setup. This strategy would provide a promising way to accelerate design-build-test cycles for metabolic engineering to improve the production of desired products.

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“…18 Although cell extracts are preserved by ultralow temperature storage (−80 °C) 34 or lyophilized for long-term storage, 20,24,35−41 aliquots without cryo-or lyo-protectants remain capable of consistent protein synthesis yields after 5 freeze−thaw cycles. 42 However, to our knowledge, there is not clear evidence on the consistency of thawed extracts and on how long extracts can be safely stored, especially while maintaining high metabolic activity. A rigorous analysis of the impact of prolonged storage on cell-free metabolism would address these open questions.…”

Section: ■ Introductionmentioning

confidence: 99%

Cell Extracts from Bacteria and Yeast Retain Metabolic Activity after Extended Storage and Repeated Thawing

et al. 2023

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Cell-free synthetic biology enables rapid prototyping of biological parts and synthesis of proteins or metabolites in the absence of cell growth constraints. Cell-free systems are frequently made from crude cell extracts, where composition and activity can vary significantly based on source strain, preparation and processing, reagents, and other considerations. This variability can cause extracts to be treated as black boxes for which empirical observations guide practical laboratory practices, including a hesitance to use dated or previously thawed extracts. To better understand the robustness of cell extracts over time, we assessed the activity of cell-free metabolism during storage. As a model, we studied conversion of glucose to 2,3-butanediol. We found that cell extracts from Escherichia coli and Saccharomyces cerevisiae subjected to an 18-month storage period and repeated freeze−thaw cycles retain consistent metabolic activity. This work gives users of cell-free systems a better understanding of the impacts of storage on extract behavior.

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<em>Escherichia coli</em>-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Cited by 16 publications

References 0 publications

At-home, cell-free synthetic biology education modules for transcriptional regulation and environmental water quality monitoring

At-home, cell-free synthetic biology education modules for transcriptional regulation and environmental water quality monitoring

Rapid prototyping enzyme homologs to improve titer of nicotinamide mononucleotide using a strategy combining cell‐free protein synthesis with split GFP

Cell Extracts from Bacteria and Yeast Retain Metabolic Activity after Extended Storage and Repeated Thawing

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