Plasmid incompatibility: more compatible than previously thought?

It is generally accepted that plasmids containing the same origin of replication are incompatible. We have re-examined this concept in terms of the plasmid copy number, by introducing plasmids containing the same origin of replication and different antibiotic resistance genes into bacteria. By selecting for resistance to only one antibiotic, we were able to examine the persistence of plasmids carrying resistances to other antibiotics. We find that plasmids are not rapidly lost, but are able to persist in bacteria for multiple overnight growth cycles, with some dependence upon the nature of the antibiotic selected for. By carrying out the experiments with different origins of replication, we have been able to show that higher copy number leads to longer persistence, but even with low copy plasmids, persistence occurs to a significant degree. This observation holds significance for the field of protein engineering, as the presence of two or more plasmids within bacteria weakens, and confuses, the connection between screened phenotype and genotype, with the potential to wrongly assign specific phenotypes to incorrect genotypes.     

Characteristics of different nucleic acid staining dyes for DNA fragment sizing by flow cytometry

An efficient and reliable double-stranded DNA (dsDNA) staining protocol for DNA fragment sizing by flow cytometry is presented. The protocol employs 0.8 microM of PicoGreen to label a wide range of DNA concentrations (0.5 ng/mL to 10,000 ng/mL) without regard to the solution dye/bp ratios and without initial quantification of the DNA analyte concentration. Using a combination of spectrofluorometry and flow cytometry experiments, we found that PicoGreen exhibited better overall performance than all the tested dsDNA binding dyes, such as TOTO-1. Fluorometric titration revealed that typical DNA staining protocols designed on the basis of the dye/bp ratio were highly dependent upon the DNA concentration for optimal results. PicoGreen was the least sensitive to the solution dye/bp ratio and was highly fluorescent in the presence of dsDNA. Using this new protocol, accurate histograms of HindIII digested lambda DNA were demonstrated for DNA concentrations ranging from 5 to 2000 ng/mL, and for dye/bp ratios from 106:1 to 1:4 at 0.8 microM of PicoGreen. The new one-step protocol is broadly applicable to any sensitive, laser-induced fluorescence method for detection of nucleic acids.     

Using T7 phage display to select GFP-based binders

Filamentous phage do not display cytoplasmic proteins very effectively. As T7 is a cytoplasmic phage, released by cell lysis, it has been prospected as being more efficient for the display of such proteins. Here we investigate this proposition, using a family of GFP-based cytoplasmic proteins that are poorly expressed by traditional phage display. Using two single-molecule detection techniques, fluorescence correlation spectroscopy and anti-bunching, we show that the number of displayed fluorescent proteins ranges from one to three. The GFP derivatives displayed on T7 contain binding loops able to recognize specific targets. By mixing these in a large background of non-binders, these derivatives were used to optimize selection conditions. Using the optimal selection conditions determined in these experiments, we then demonstrated the selection of specific binders from a library of GFP clones containing heavy chain CDR3 antibody binding loops derived from normal donors inserted at a single site. The selected GFP-based binders were successfully used to detect binding without the use of secondary reagents in flow cytometry, fluorescence-linked immunosorbant assays and immunoblotting. These results demonstrate that specific GFP-based affinity reagents, selected from T7-based libraries, can be used in applications in which only the intrinsic fluorescence is used for detection.     

Novel iminocoumarin dyes as photosensitizers for dye-sensitized solar cell

Novel iminocoumarin dyes (2a-c and 3a-c) having carboxyl and hydroxyl anchoring groups onto the dyes skeletons have been designed and synthesized for the application of dye-sensitized nanocrystalline TiO₂ solar cells (DSSCs). The photophysical and electrochemical studies showed that these iminocoumarin dyes are suitable as light harvesting sensitizers in DSSC application. The dyes having carboxyl and hydroxyl anchoring groups (2a-c) showed better efficiency when compared to the dyes having carboxyl group (3a-c) alone. The cell consisted of dye 2a generated the highest solar-to-electricity conversion efficiency (η) of 0.767% (open circuit voltage (V_oc) = 0.491 V, short circuit photocurrent density (J_sc) = 2.461 mA cm⁻², fill factor (ff) = 0.635) under simulated AM 1.5 irradiation (1000 W m⁻²) with a total semiconductor area of 0.25 cm². The corresponding incident photon-to-current conversion efficiency (IPCE) of the above cell was 21.38%. The overall low efficiency of the dyes is ascribed to the lack of light harvesting ability at longer wavelength region.