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Article Dans Une Revue Journal of visualized experiments : JoVE Année : 2022

Cell-Free protein synthesis from exonuclease-deficient cellular extracts utilizing linear DNA templates

Résumé

Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA templates for CFPS will further enable the technology to reach its full potential, decreasing the experimental time by eliminating the steps of cloning, transformation, and plasmid extraction. Linear DNA can be rapidly and easily amplified by PCR to obtain high concentrations of the template, avoiding potential in vivo expression toxicity. However, linear DNA templates are rapidly degraded by exonucleases that are naturally present in the cell extracts. There are several strategies that have been proposed to tackle this problem, such as adding nuclease inhibitors or chemical modification of linear DNA ends for protection. All these strategies cost extra time and resources and are yet to obtain near-plasmid levels of protein expression. A detailed protocol for an alternative strategy is presented here for using linear DNA templates for CFPS. By using cell extracts from exonuclease-deficient knockout cells, linear DNA templates remain intact without requiring any end-modifications. We present the preparation steps of cell lysate from Escherichia coli BL21 Rosetta2 ΔrecBCD strain by sonication lysis and buffer calibration for Mg-glutamate (Mg-glu) and K-glutamate (K-glu) specifically for linear DNA. This method is able to achieve protein expression levels comparable to that from plasmid DNA in E. coli CFPS.

Domaines

Biotechnologies
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JoVE_main.pdf (231.26 Ko) Télécharger le fichier
JoVE_LinDNA_Tables_1-9.zip (103.66 Ko) Télécharger le fichier
JoVE_Materials-64236_MK2_2022-08-01.xlsx (75.12 Ko) Télécharger le fichier
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JoVE_SupFile_1_TXTL_Linear_Plasmid_calibration.xlsx (32.58 Ko) Télécharger le fichier
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Dates et versions

hal-03748754 , version 1 (10-08-2022)

Identifiants

  • HAL Id : hal-03748754 , version 1
  • DOI : 10.3791/64236
  • WOS : 000927873000071

Citer

Mahnaz Sabeti Azad, Angelo Cardoso Batista, Jean-Loup Faulon, Chase L Beisel, Jérôme Bonnet, et al.. Cell-Free protein synthesis from exonuclease-deficient cellular extracts utilizing linear DNA templates. Journal of visualized experiments : JoVE, inPress, ⟨10.3791/64236⟩. ⟨hal-03748754⟩
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