Rt Qpcr Experiment Total Rna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "A versatile active learning workflow for optimization of genetic and metabolic networks"
Article Title: A versatile active learning workflow for optimization of genetic and metabolic networks
Figure Legend Snippet: Application of METIS for optimization of a LacI gene circuit. a) Single and multi-level controller LacI gene circuits from Greco et al . 43 Characterization of these circuits through dynamic range (DR) and/or fold-change (FC) of the output (Gfp fluorescence) between 0 and 10 mM input (concentration of IPTG). b) Imported in the active learning notebook, the varied components of the reactions included 4 lacI circuits as alternatives, some factors of buffer and energy mix of E. coli cell-free system along with the lysate, as well as T7 RNA polymerase and a second plasmid expressing lacI under a T7 promoter. c) The average of triplicates as the result of 10 rounds of active learning as plots for the objective function (FC × DR) and fold change (FC) values. d) Plots showing the distribution of measured yield values within the ranges of each factor. e) Feature importance percentages showing the effect of each factor on decision-making by the model to predict objective function values. f) Titration of P T7 - LacI plasmid and T7 RNA polymerase with the optimal composition (from 10 rounds of active learning that achieved with pTHS LacI circuit, the toehold switch as the second level controller of gene expression through translation). The heatmaps show FC × DR (left) and FC (right) values (average of triplicates) of the titration. g) The same titration experiment as in (f) but instead of the pTHS circuit, Gfp was expressed under a constitutive promoter (independent from the P T7 - LacI plasmid and T7 RNA polymerase on the protein production). h) Titration (0, 1, 3, 10, 30, and 100 nM) of LacI plasmids with either constitutive or T7 promoter in combination with 10 nM of a Gfp plasmid (with T7 or constitutive promoter). i) The RT-qPCR results of the relative level of LacI and Gfp mRNAs for a similar experiment in (h) (0, 10, 100 nM LacI plasmids, and 10 nM Gfp plasmids) after 10 hours. Relative log2 resource share between LacI and Gfp mRNA in each sample is reported in order to account for RNA purification efficiency variability j) The 20 most informative combinations were downloaded after the 10-round active learning and the P T7 - LacI plasmid with purified LacI were replaced. After performing the experiments and measuring the objective function, we imported them as Day 0 and continued with experiments of the next round’s predictions (Day 1). k) Plots of the objective function FC × DR (left) and FC (right) values (average of triplicates) of 20 most informative combinations with purified LacI followed by Day 1 experiments suggested by the workflow. See Data availability for combinations and objective function values.
Techniques Used: Fluorescence, Concentration Assay, Plasmid Preparation, Expressing, Titration, Quantitative RT-PCR, Purification