Journal: bioRxiv

Article Title: Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system

doi: 10.1101/099838

Figure Lengend Snippet: Comparison of transcription and translation between PURE system and RTS 100 E. coli HY S30 cell extract system. A. RNA denaturing gel of PURE reaction transcribing TC-Fluc mRNA. PURE system with no plasmid template and plasmid encoding TC-Fluc were incubated at 37°C for 2 h. Total RNA were purified by phenol/chloroform extraction and isopropanol precipitation from equal volumes of the two reactions and then run on lane 2 and 3, respectively. B. RNA denaturing gel of S30 extract transcribing TC-Fluc mRNA. S30 extract with no plasmid template and plasmid encoding TC-Fluc were incubated at 30°C for 6 h. Total RNAs were purified by phenol/chloroform extraction and isopropanol precipitation from equal volumes of the two reactions and then run on lane 2 and 3, respectively. C. Assessment of active TC-Fluc and TC-Fluc-chis proteins translated in PURE system and S30 extract. Equal volume aliquots were taken for luciferase assay to measure the actual amount of active TC-Fluc produced. Values represent averages and error bars are ± standard deviations, with n=3. D. Assessment of total TC-Fluc and TC-Fluc-chis produced in PURE system and S30 extract. TC-Fluc and TC-Fluc-chis synthesized in C were incubated with FlAsH-EDT 2 biarsenical labeling reagent and analyzed on a SDS-PAGE. The gel was scanned by a typhoon scanner with filter set (508nmE×/528nmEm). Asterisk * indicates full lenth product.

Article Snippet: RTS 100 E.coli HY Kit (S30 system) was purchased from 5PRIME.

Techniques: Plasmid Preparation, Incubation, Purification, Luciferase, Produced, Synthesized, Labeling, SDS Page

Journal: bioRxiv

Article Title: Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system

doi: 10.1101/099838

Figure Lengend Snippet: Assessment of PURE and RTS 100 E. coli HY S30 system translation by production of HaloTag fusion proteins using linear DNA templates. HT-Ch-TolA, HT-EntE-Ch-TolA and HT-EntF-Ch-TolA with and without stop codon (90 kD, 160 kD, 220 kD) were synthesized in the PURE system and S30 extract and then incubated with HaloTag TMR Ligand. Equal volume aliquots of samples from PURE system and S30 extract reactions were analyzed on a SDS-PAGE and then scanned by a typhoon scanner with filter set (555nmE×/580nmEm). Lane 1, 3, 5, 8, 10, 12 are HaloTag fusion proteins with stop codon. Lane 2, 4, 6, 9, 11, 13 are HaloTag fusion proteins without stop codon. Lane 1 and 14 are negative controls with no template. (HT: HaloTag; Ch: mCherry. TolA is a C-terminal 171-amino-acid alpha-helical spacer excised from E. coli TolA domain II. EntE and EntF are multidomain enzymes from E. coli enterobactin biosynthetic pathway.)

Article Snippet: RTS 100 E.coli HY Kit (S30 system) was purchased from 5PRIME.

Techniques: Synthesized, Incubation, SDS Page