reactions cell free transcription translation systems  (New England Biolabs)

Journal: bioRxiv

Article Title: Antitoxin-induced auto-phosphorylation neutralizes the nucleotidyltransferase toxin AbiEii from Streptococcus agalactiae to safeguard global translation

doi: 10.64898/2026.01.19.700257

Figure Lengend Snippet: T65 is important, but not essential, for NTase and phosphorylation activity. ( A ) In vitro transcription/translation reactions assessing levels of DHFR control protein produced in the presence of either 5 µM AbiEii WT or AbiEii-p. ( B ) In vitro transcription/translation reactions assessing levels of DHFR control protein produced in the absence and presence of 5 µM AbiEii T65A, AbiEii D192A, or AbiEii-p. Reactions were loaded onto 4-20% gradient gels ran at 180 V for 1 h. ( C ) Densitometric analysis of SDS-PAGE bands from ( B ) corresponding to DHFR. ( D ) Dose-response curves of increasing concentrations of either AbiEii WT or AbiEii T65A plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4A and B . ( E ) Logarithmic dose-response curves of either 5 µM AbiEii WT or AbiEii T65A incubated with increasing concentrations of AbiEi plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4C and D . Logarithmic curves were generated using non-linear regression in Prism (GraphPad). ( F ) Endpoint viable count antitoxicity assays of E. coli DH5α transformed with either pBAD30 empty vector, pTRB482 (AbiEii WT), or pTRB736 (AbiEii T65A), and either pTA100 empty vector or pTRB481 (AbiEi). Overnight cultures were re-seeded into fresh LB supplemented with Ap, Sp and D-glu and grown to mid-log phase. Samples were serially diluted 10 −2 -10 −9 and spotted onto M9A plates containing Ap and Sp, with or without D-glu, L-ara, and IPTG for repression of toxin expression, induction of toxin expression, and induction of antitoxin expression, respectively. Plates were incubated at 37 °C for 48 h, after which they were imaged and colonies counted to determine CFU/ml (one-way ANOVA, P < 0.05). “T” = toxin induced, “TA” = antitoxin + toxin induced. Assays shown are representative of three independent biological replicates and plotted data represent the mean ± SEM.

Article Snippet: Five μM of purified AbiEii WT or AbiEii-p were added to cell-free transcription/translation protein synthesis reactions (PURExpress ® ; NEB) to quantify changes in DHFR control protein expression.

Techniques: Phospho-proteomics, Activity Assay, In Vitro, Control, Produced, SDS Page, Expressing, Generated, Incubation, Transformation Assay, Plasmid Preparation