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...

A pair of primers were designed according to the nsp5 gene of human group B rotavirus strain WH-1, and the nsp5 gene was amplified by PCR and subcloned into the expression vector pGEX-KG. After induced with IPTG, the NSP5 was expressed as a GST fusion protein in the soluble form. Then the protein was purified and immunized mice to obtain specific antiserum. At last, polyclonal antibody was anal...

ColE1 derivative plasmids were constructed in which the natural promoter that primes replication or, in addition, the region coding for the RNA I control element had been deleted. In all of these molecules priming of the origin was effected by read-through transcription from constitutive or inducible (lacUV5) promoters inserted farther upstream. In the latter case, regulation of lac repressor a...

UreG is one of the accessory proteins of urease in plants. In this study, a gene encoding an UreG consisting of 363 amino acids from Arabidopsis thaliana was isolated. The AtUreG gene was expressed in Escherichia coli BL21 as a GST fusion protein. The GST-AtUreG fusion protein was induced and purified under the optimized culturing conditions of 0.2 mM IPTG and 25°C for 5 h. Western blot analysi...

The field of synthetic biology has produced genetic circuits capable of emulating functional paradigms seen in digital electronic circuits. Examples are bistable switches, oscillators, and logic gates. The present work combines detailed mechanistic-kinetic models and stochastic simulation techniques as well as the techniques of in vivo molecular biology to study the potential of a synthetic, si...

In this work we present cloning and overexpression of lactococcal CcpA protein in Escherichia coli Xl1blue strain as a fusion with 6 x His tag. A high yield of the CcpA protein was obtained when the cells were cultured in liquid medium LB with 100 microg/ml ampicillin at 37 degrees C and subsequently for 4 h after induction by IPTG. The procedure let us obtain 5 mg of homogenous CcpA protein. G...

Indolicidin is an antimicrobial cationic peptide naturally present in the bovine neutrophils. In this study, transformed Escherichia coli cells were employed for greater and cost effective production of indolicidin. Four strains of E. coli [C41 (DE3), C43 (DE3), C41 (DE3) pLysS & C43 (DE3) pLysS] were transformed with recombinant plasmid pET21A carrying the indolicidin gene and were successfull...

No. bell9220 Beamline(s): X25 Introduction: In previous work, crystal structures of the E. coli Lac repressor bound to operator and to the inducer isopropylthiogalactoside (IPTG) provided a model for how the binding of the inducer reduces the affinity of the repressor for operator. However, because of the low resolution of the operator-bound structure (4.8 Å), the model for the allosteric trans...

The kinetics of transcription initiation in Escherichia coli depend on the duration of two rate-limiting steps, the closed and the open complex formation. In a lac promoter variant, P(lac/ara-1), the kinetics of these steps is controlled by IPTG and arabinose. From in vivo single-RNA measurements, we find that induction affects the mean and normalized variance of the intervals between consecuti...

The cDNA encoding the precursor form of non-specific lipid-transfer protein (pre-nsL-TP) from rat liver was cloned into the expression vector pET3d. The resulting plasmid was transformed to the Escherichia coli strain BL21(DE3). After induction of the bacteria with isopropyl-beta-D-thiogalactopyranoside (IPTG) pre-nsL-TP was purified from the bacterial lysate by anion exchange chromatography fo...