Light can be used to control numerous cellular processes including protein function and interaction as well as gene expression in a non-invasive fashion and with unprecedented spatiotemporal resolution. However, for chemical phototriggers tight, gradual, and homogeneous light response has never been attained in living cells. Here, we report on a light-responsive bacterial T7 RNA polymerase expr...

Conditional lethal mutations are valuable for analyzing essential genes. We describe here a derivative of the bacterial transposon Tn5 called Tn5tac1 and its use in an innovative strategy for making mutations with conditional phenotypes. The 4.6-kilobase Tn5tac1 element contains a strong, regulatable, outward-facing promoter (Ptac) near one end and is polar on the expression of distal genes whe...

We have studied the lac repressor (lacR) system in two breast cancer cell lines, MCF-7 and MDA-MB-231, in vitro and in vivo. Breast cancer cell lines were stably transfected with lacR and tested for inducibility by transient transfection with a lac operator/luciferase reporter plasmid. The level of expression of lacR did not appear to correlate with the basal or maximal activation of induction ...

Recombinant heterologous proteins can be produced as insoluble aggregates partially or perfectly inactive in Escherichia coli. One of the strateges to improve the solubility of recombinant proteins is fusion with a partner that is excellent in producing soluble fusion proteins. To improve the production of soluble β-galactosidase, the gene of Thermus thermophilus KNOUC112 β-galactosidase (KNOUC...

Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (P(BAD)) system that is more compatible with IPTG (isopropyl-beta-D-1-thiogalactopyranoside) induct...

Growth and media Liquid cultures were grown at 37°C with aeration in minimal A medium (Miller, 1992) with 0.2% glycerol or glucose as the carbon source or in LB Miller medium (Difco). Colonies were grown at 37°C on 1.5% agar plates made from LB Miller medium or minimal A medium with glycerol as the carbon source. Plasmids were maintained by supplementing media with 50 μg/mL of ampicillin, 15 μg...

The aim of the study was to optimize conditions for producing Salmonella Enteritidis recombinant heat shock protein 60 (rHsp60). Seven Escherichia coli host strains (Rosetta, Turner, C41, C43, Origami, BL21pLys, Rosetta pLys) were transformed by a recombinant plasmid containing Hsp60 gene from Salmonella Enteritidis, and then cultured and induced by isopropyl-beta-D-thiogalactopyranoside (IPTG)...

Alkaline phosphatase is an enzyme with widespread use in research and industry such as protein labeling, dephosphorylation of nucleic acids, and enzyme based biosensors. In the present study, alkaline phosphatase gene was inserted into the pET15b vector. The recombinant DNA was then expressed using IPTG as an inducer. The expression of this enzyme was optimized by changing expression conditions...

Induction parameters including inducer concentration, period of induction and the cell concentration at which inducer is to be added to the fermentation broth were optimized in order to increase the yield of the EcoRI restriction endonuclease isolated from recombinant E. coli. Bacterial cells harboring the plasmid pPG430 containing EcoRI endonuclease and the methylase genes under the control of...