Directed evolution of nucleotide libraries using recombination or mutagenesis is an important technique for customizing catalytic or biophysical traits of proteins. Conventional directed evolution methods, however, suffer from cumbersome digestion and ligation steps. Here, we describe a simple method to increase nucleotide diversity using single-stranded DNA (ssDNA) as a starting template. An initial PCR amplification using phosphorylated primers with overlapping regions followed by treatment with lambda exonuclease generates ssDNA templates that can then be annealed via the overlap regions. Double-stranded DNA (dsDNA) is then generated through extension with Klenow fragment. To demonstrate the applicability of this methodology for directed evolution of nucleotide libraries, we generated both gene shuffled and regional mutagenesis synthetic antibody libraries with titers of 2×108 and 6×107, respectively. We conclude that our method is an efficient and convenient approach to generate diversity in nucleic acid based libraries, especially recombinant antibody libraries.
Laboratories intending to adopt cycle sequencing of PCR products in their routine analysis often face a confusing range of methods and kits. Through the study of mitochondrial cytochrome b, we have shown that clean and highly reproducible sequences could be obtained by using a combination of existing simple and economical methods in the preparation of DNA templates, PCR, purification of PCR products and sequencing. Our protocol is useful in itself or as a standard in typing other PCR-amplified DNA at the population level.
A filamentous phage bearing the peptide sequence TLTTKLY was isolated from a heptapeptide phage display library against a velogenic Newcastle disease virus (NDV). In order to investigate the potential of this specific phage as an immunological reagent in virus pathotyping, an enzyme-linked immunosorbent assay (ELISA)-based method was developed. This method can differentiate the velogenic strains from the mesogenic and lentogenic strains. An equilibrium-binding assay in solution showed that the interactions between the phage and all the NDV strains gave rise to two widely differing dissociation constants (Kdrel). Based upon the first Kdrel values, NDV strains can be classified into two groups; the first comprises the velogenic strains, and the second consists of the mesogenic and lentogenic strains. These results indicate a high degree of correlation between the binding affinities and pathotyping of NDV strains using the TLTTKLY phage.
Endolysins have been studied intensively as an alternative to antibiotics. In this study, endolysin derived from a phage which infects methicillin-resistant Staphylococcus aureus (MRSA) was cloned and expressed in Escherichia coli pET28a. Initially, the endolysin was cloned using BamHI/XhoI, resulting in expression of a recombinant endolysin which was expressed in inclusion bodies. While solubilization was successful, the protein remained nonfunctional. Recloning the endolysin using NcoI/XhoI resulted in expression of soluble and functional proteins at 18°C. The endolysin was able to form halo zones on MRSA plates and showed a reduction in turbidity of MRSA growth. Therefore, cloning strategies should be chosen carefully even in an established expression system as they could greatly affect the functionality of the expressed protein.
Gene assembly methods are an integral part of molecular cloning experiments. The majority of existing vector assembly methods stipulate a need for exonucleases, endonucleases and/or the use of single-stranded DNA as starting materials. Here, we introduced a vector assembly method that employs conventional PCR to amplify stable double-stranded DNA fragments and assembles them into functional vectors specifically for antibody chain shuffling. We successfully formed vectors using cassettes amplified from different templates and assembled an array of single chain fragment variable clones of fixed variable heavy chain, with different variable light chains - a chain shuffling process for antibody maturation. The method provides an easy alternative to the conventional cloning process.
We describe a novel automated cell detection and counting software, QuickCount® (QC), designed for rapid quantification of cells. The Bland-Altman plot and intraclass correlation coefficient (ICC) analyses demonstrated strong agreement between cell counts from QC to manual counts (mean and SD: -3.3 ± 4.5; ICC = 0.95). QC has higher recall in comparison to ImageJauto, CellProfiler and CellC and the precision of QC, ImageJauto, CellProfiler and CellC are high and comparable. QC can precisely delineate and count single cells from images of different cell densities with precision and recall above 0.9. QC is unique as it is equipped with real-time preview while optimizing the parameters for accurate cell count and needs minimum hands-on time where hundreds of images can be analyzed automatically in a matter of milliseconds. In conclusion, QC offers a rapid, accurate and versatile solution for large-scale cell quantification and addresses the challenges often faced in cell biology research.
Reverse genetics has been used to generate recombinant Newcastle disease virus with enhanced immunogenic properties for vaccine development. The system, which involves co-transfecting the viral antigenomic plasmid with three helper plasmids into a T7 RNA polymerase-expressing cell to produce viral progenies, poses a great challenge. We have modified the standard transfection method to improve the transfection efficiency of the plasmids, resulting in a higher titer of virus progeny production. Two transfection reagents (i.e., lipofectamine and polyethylenimine) were used to compare the transfection efficiency of the four plasmids. The virus progenies produced were quantitated with flow cytometry analysis of the infectious virus unit. The modified transfection method increased the titer of virus progenies compared with that of the standard transfection method.