Abbreviations: CAPS: N-cyclohexyl-3-aminopropanesulfonic acid cDNA: omplementary DNA to mRNA CHES: N-cyclohexyl-2-aminoethanesulfonic acid. DTT: dithiothreitol FPLC: fast protein liquid chromatography IPTG: isopropylthio-β-D-galactoside LB: Luria-Bertani MES: 2-(N-morpholino)ethanesulfonic acid Ni-NTA: Nickel-nitriloacetic acid ORF: open reading frame PCR: polymerase chain reaction PMSF: phenyl...

RepA, the replication initiator protein of the IncB plasmid pMU720, acts preferentially in cis. The cis activity of RepA is thought to be mediated by CIS, a 166-bp region of DNA separating the coding region of repA from the origin of replication (ori) of pMU720. To investigate the trans activity of RepA, the repA gene, without its cognate ori, was cloned on a multicopy plasmid, pSU39. The ori o...

Riboswitches are RNA-based regulatory devices that mediate ligand-dependent control of gene expression. However, there has been limited success in rationally designing riboswitches. Moreover, most previous riboswitches are confined to a particular gene and only perform one-way regulation. Here, we used a library screening strategy for efficient creation of ON and OFF riboswitches of lacI on the...

We have developed a fast and accurate method to engineer the Bacillus subtilis genome that involves fusing by PCR two flanking homology regions with an antibiotic resistance gene cassette bordered by two mutant lox sites (lox71 and lox66). The resulting PCR products were used directly to transform B. subtilis, and then transient Cre recombinase expression in the transformants was used to recomb...

In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in...

By taking advantage of MazF, an ACA codon-specific mRNA interferase, Escherichia coli cells can be converted into a bioreactor producing only a single protein of interest by using an ACA-less mRNA for the protein. In this single-protein production (SPP) system, we engineered MazF by replacing two tryptophan residues in positions 14 and 83 with Phe (W14F) and Leu (W83L), respectively. Upon the a...