Journal: bioRxiv

Article Title: Rapid, Low-Cost Detection of Water Contaminants Using Regulated In Vitro Transcription

doi: 10.1101/619296

Figure Lengend Snippet: a , T7 RNA polymerase transcription of the fluorescence-activating three-way junction dimeric Broccoli (3WJdB) aptamer. Binding of DFHBI-1T by 3WJdB activates its fluorescence. b , Titration of the linear dsDNA transcription template in a commercially-available transcription kit generates micromolar amounts of mean equivalent fluorescence (MEF) when standardized to soluble fluorescein isothiocyanate (µM FITC) measured under identical plate reader conditions. c , Visual detection of 3WJdB:DFHBI-1T fluorescence, with values corresponding to MEF of soluble FITC. d , Comparison of fluorescence kinetics using an equimolar concentration of DNA template (50 nM) encoding either 3WJdB in a commercially available transcription kit or sfGFP in commercially-available transcription-translation kit. All data shown for 3 experimental replicates as individual lines with raw fluorescence values standardized to MEF (µM FITC). Shading indicates the average value ± standard deviation.

Article Snippet: Plasmids encoding transcription templates were constructed using pUC19-T7-3WJdB-T (Addgene plasmid #87308) as a backbone.

Techniques: Fluorescence, Binding Assay, Titration, Concentration Assay, Standard Deviation

Journal: bioRxiv

Article Title: Rapid, Low-Cost Detection of Water Contaminants Using Regulated In Vitro Transcription

doi: 10.1101/619296

Figure Lengend Snippet: In vitro transcriptions can be allosterically regulated by designing a synthetic transcription template configured to bind a purified transcription factor (TetR) via the operator sequence ( tetO ) placed downstream of the T7 promoter. When bound by TetR, the aTF serves as a steric block to transcription elongation. a , Spacing between the T7 promoter and tet O site influences the ability to regulate transcription in the presence of TetR. A series of spacers in 2 base pair (BP) intervals was constructed to evaluate the impact of spacer length on the ability to regulate transcription. b , End point data shown for promoter-operator spacer variants regulated (with 2.5 µM TetR dimer, 100-fold excess over DNA template) and unregulated (without TetR) after 240 minutes. c , Kinetic comparison of 3WJdB fluorescence activation of TetR-regulated versus unregulated transcription reactions with a 4 BP spacer. d , Induction of a TetR regulated in vitro transcription reaction occurs in the presence of the cognate ligand, anhydrotetracycline (aTc), by binding to TetR and preventing its binding to the DNA template. This allows transcription to proceed, leading to fluorescence activation. e , Dose response with anhydrotetracycline (aTc), measured at 240 minutes with 25 nM DNA and 1.25 µM TetR dimer. f , Kinetics of induction (from e ) with 2.5 µM aTc. All data shown for 3 experimental replicates as lines or points with raw fluorescence values standardized to MEF (µM FITC). Shading ( c, f ) and error bars ( e ) indicate the average value of experimental replicates ± standard deviation.

Article Snippet: Plasmids encoding transcription templates were constructed using pUC19-T7-3WJdB-T (Addgene plasmid #87308) as a backbone.

Techniques: In Vitro, Purification, Sequencing, Blocking Assay, Construct, Fluorescence, Activation Assay, Binding Assay, Standard Deviation

Journal: bioRxiv

Article Title: Rapid, Low-Cost Detection of Water Contaminants Using Regulated In Vitro Transcription

doi: 10.1101/619296

Figure Lengend Snippet: a , A kleptamer RNA strand (KB2) antisense to the dye-binding region of the broccoli aptamer can disrupt folding and lead to the loss of fluorescence. Addition of a KB2-expressing template in a 4:1 ratio with the 3WJdB template inhibits signal. b , Kleptamers can be used to invert the response of transcription factors. KB2 transcription can be regulated by TetR (1.25 μM dimer) and aTc (2.5 μM shown) by placing the tetO site in between the T7 promoter and KB2 coding sequence. c , This scheme was used to create a ROSALIND zinc sensor using the aporepressor AdcR. When bound to Zn 2+ (30 μM), AdcR (1.5 μM dimer) binds to its cognate operator sequence, adcO , placed upstream of the KB2 coding sequence, preventing KB2 from being expressed and thereby activating fluorescence. Arrows inside of the plots represent direction of regulation when indicated species are added. All data shown for 3 experimental replicates as lines with raw fluorescence values standardized to MEF (µM FITC). Shading indicates the average value ± standard deviation. 3WJdB DNA template concentrations used are: 25 nM for a, b and 7.5 nM for c . KB2 DNA template concentrations used are: 100 nM for a , and 150 nM for b, c .

Article Snippet: Plasmids encoding transcription templates were constructed using pUC19-T7-3WJdB-T (Addgene plasmid #87308) as a backbone.

Techniques: Binding Assay, Fluorescence, Expressing, Sequencing, Standard Deviation