Journal: Genes

Article Title: Development of Toehold Switches as a Novel Ribodiagnostic Method for West Nile Virus

doi: 10.3390/genes14010237

Figure Lengend Snippet: In vitro trials of WNV toehold switches ( A ) Individual in vitro Transcription/Translation (IVT-TA) trials of thirty selected GFP-toeholds of WNV. Signal detection was performed in four different channels, corresponding to 507 (blue), 509 (green), 511 (yellow) and 514 (orange) nm across the GFP emission spectrum. Y-axis shows fold enrichment of GFP detection compared to the average of negative controls. Negative controls include IVT-TA trials of different toeholds mixes in the absence of trigger RNA and IVT-TA without any WNV toehold in the presence of 100 ng WNV RNA. ( B ) Independent biological repetitions of the selected toeholds using 100 ng of WNV lineage 2 genomic RNA trigger ( C ) IVT-TA trials of mixed WNV toeholds using both lineage 1 and 2 isolated RNA as trigger. In addition, T10284 that outperformed all other toeholds was also tested individually against both lineage triggers.

Article Snippet: In vitro Transcription-Translation (IVT-TA) reactions were performed with PURExpress® kit (NEB #E6800L, Bioline Thessaloniki, Greece), as previously described [ ], in the presence of 100ng WNV cultured S/N RNA unless otherwise stated in text.

Techniques: In Vitro, Isolation

Journal: bioRxiv

Article Title: Shield co-opts an RmuC domain to mediate phage defence across Pseudomonas species

doi: 10.1101/2022.11.04.515146

Figure Lengend Snippet: (a) Cells carrying plasmid pBAD18 encoding ShdA II-His6 were grown for 5 hrs in the presence of 0.2 % L-arabinose. Cells were fractionated to produce soluble and membrane samples and analysed by immunoblot with antibodies to the His6 tag, GroEL (cytoplasmic control) and TatA (membrane control). (b-e) In vitro DNAse activity assays using (b) ShdA II 138-524 (c) ShdA I 140-526 (d) ShdA III 135-523 and (e) ShdA IV 135-521 . ShdA proteins and DHFR were synthesised using the cell-free PURExpress kit (NEB). DNAse activity was tested against 10 ng of input DNA. DNA types tested were phage DNA, E. coli MG1655 chromosomal DNA and plasmid (pSG483) DNA. For ShdA I 140-526 , ShdA III 135-523 and ShdA II 138-524 phage DNA was from ϕSipho. For ShdA IV 135-521 phage DNA was from ϕAlma.

Article Snippet: The RmuC domains of ShdA I, ShdA II, ShdA III and ShdA IV homologues were synthesised in vitro using the PURExpress cell-free transcription/translation kit (NEB), from PCR products.

Techniques: Plasmid Preparation, Western Blot, In Vitro, Activity Assay