Among these clones, AcPheRS-8G and AcPheRS-12B displayed the fastest growth rate in the presence of chloramphenicol and the brightest GFP fluorescence (The selection plasmid, pREP, contains a T7 RNA polymerase gene with amber mutation at permissive site. The synthesis of full-length T7 RNA polymerase with amber suppression drives the expression of a green fluorescent protein).
The success of the new approach likely hinges on the inherent strength of the expression system since previous attempts to utilize fluorescence-based selection used two [ 12] or more (up to 5) [ 13] rounds of FACS selection.
The developed UMT reporter system is sensitive for monitoring protein solubility based on coupled fluorescence and chemical selection.
Other screening strategies are based on spectrophotometric analyses of colored by-products, fluorescence applications, growth selection and, to a minor extent, the use of biosensors indicating the pool of isoprenoid precursors.
Stable genetic modification of hMSC following plasmid DNA electroporation was performed by culture under combined antibiotics selection, fluorescence activated cell sorting (FACS) and/or single cell cloning [ 41].
Generally, sampling cells from more extreme tails by decreasing the fluorescence cutoff for selection increased power; however, mutations causing very large increase or decrease in the standard deviation of the fluorescence phenotype were found to require a particular range of cutoff percentages to maximize power.
The population distribution of log-fluorescence levels after selection is then (75) p ′ (x ) = ∫ d μ ρ ′ 2 π σ exp [ − (x − μ ) 2 2 σ 2 ].
Several kanamycin-resistant root lines with sGFP S65T) (synthetic green-fluorescent protein with S65T mutation) fluorescence were obtained after selection and regeneration.
To generate the final HIV-1 shRNA-library, the shRNA-sequences were subcloned by LR recombination into the lentiviral vector pLP/EGFP/shLib (Fig. 1e, final library) which allows for the enrichment of transduced cell lines by puromycin selection and fluorescence monitoring by means of enhanced green fluorescence protein (EGFP).
GCs were chosen randomly by using phase optics to avoid biased selection of fluorescence, and then a fluorescent image was captured, exposure time being kept constant and below greyscale pixel saturation.
