0-Galactosidase activity

Procedures of the beta-galactosidase activity measuring using colour reaction with ONPG and X-Gal without cells permeabilization were developed and the detection limit at the level of 4 ppb has been achieved. The influence of the foreign ions (phosphate, sulphate, carbonate et. al) was studied. 

Fig. 3. Comparison of transfection efficiencies obtained using PolyFect Reagent, a dendrimer-based transfection reagent, and a calcium phosphate-mediated procedure. COS-7 and HeLa cells were transfected in srx-weU plates with a /3-galactosidase expression plasmid using the appropriate protocol. For the calcium phosphate-mediated transfection, 6 pg of plasmid DNA was used and the medium was changed after 5 h incubation. Transfections were performed in triplicate, and transfection efficiency was measured by monitoring the /3-galactosidase activity of extracts obtained from the transfected cells. The amoimt of /3-galactosidase activity in the extracts correlates with the transfection efficiency. Cells were harvested 48 h post-trans-fection...

Extending the emulsion to a water-in-oil-in-water mixture allowed further refinement of the IVC concept. Compartmentalization of E. coli containing semm paraoxonase variants allowed the accumulation of fluorescent product to a point where it could be detected by FACS [57]. This approach was also used with in vitro transcription and translation to evolve /3-galactosidase activity from the Ebg gene [58]. 

Fig. 6.19. Sensor CMF/J-gal to measure /J-galactosidase activity in living cells.

Sozzi GO, Trinchero GD and Fraschina AA. 1999. Controlled-atmosphere storage of tomato fruit low oxygen or elevated carbon dioxide levels alter galactosidase activity and inhibit exogenous ethylene action. J Sci Food Agric 79(8) 1056—1070. 

S-galactosidase activity (Scheme 12) (201-203). Before the enzymatic cleavage, depending on the linker, the galactopyranose or a carbonate in bidentate coordination block the water access to the Gd111 ion. After enzymatic cleavage, one water molecule enters the inner sphere and results in an increased relaxivity. 

Figure 3.7 Increase in hydrogenase and P-galactosidase activities during growth with H2 of BIO (pACI42) cells containing the hupS /acZ fusion. ONP, o-nitrophenol MB, methylene blue prot, protein solid symbols, H2 added open symbols, no H2 added.

Escherichia coli cells grown in a medium with lactose as the only carbon source are monitored for p-galactosidase activity over time with the results shown below. 

Toxi-Chromotest is a commercial toxicity assay that is based on the assessment of the inhibition of (3-galactosidase activity, measured using a chromogenic substrate and a colorimeter. A mutant strain of Escherichia coli is revitalized from a lyophilized state prior to the test [39]. The principle of the MetSoil test is similar to that of the Toxi-Chromotest . 

The MetPAD test kit (Group 206 Technologies, Gainesville, Florida) has been developed for the detection of heavy metal toxicity. It has been used to determine the toxicity of sewage water and sludge, sediments, and soil [41]. The test is based on the inhibition of (3-galactosidase activity in an Escherichia coli mutant strain. Performance of the test does not require expensive equipment and it is therefore easily applied as a field test. 

MetPLATE Kit P-galactosidase activity E. coli, inhibition of p-galactosidase activity [19,40]... 

B. W. Bainbridge, N. Mathias, R. G. Price, A. C. Richardson, J. Sandhu, and B. V. Smith, Improved methods for the detection of P-galactosidase activity in colonies of Escherichia coli using a new chromogenic substrate VBzTM-gal (2-[2-(4-P-D-galactopyranosyl-3-methoxyphenyl)-vinyl]-3-methyl-benzothiazolium toluene- 4-sulphonate), FEMS Microbiol. Lett., 80 (1991) 319-324. 

J. Langridge, Mutations conferring quantitative and qualitative increases in P-galactosidase activity in Escherichia coli, Mol. Gen. Genet., 105 (1969) 74-83. 

The characterization of the nar promoter in E. coli with the intact nar operon on the chromosome was carried out. Expression of P-galactosidase was maximal when the nar promoter was induced at ODgoo =1-7 under anaerobic conditions in the presence of 1 % nitrate. At this optimal condition the induction ratio was approximately 250 and the specific P-galactosidase activity was approximately 7500 Miller units at ODgoo = 2.7 [35]. In this study, we used plasmid... 

The maximum specific (3-galactosidase activities were also strongly dependent on the DO levels before and after induction. Maintenance of high DO levels before induction and low DO levels during the induction period was important to maximize the expression of (3-galactosidase (Table 2, rows C, E, F, and G). 

In the absence of nitrate, the maximum specific (3-galactosidase activity became only 4000 Miller units (Table 2, row H). Therefore, addition of nitrate into the growth medium before induction is necessary to maximize the expression of (3-galactosidase. 

Of three cases of different cell densities (ODgoo = 18, 35, 80) at the induction time, the highest value of specific P-galactosidase activity was obtained when... 

Third, one modified nar promoter (pMW618),whose expression is known to be independent of nitrate, was tested in different E. coli strains having narL mutation to make pMW618 more independent of nitrate. One combination, pMW618/W31 lOnarL, had excellent properties to be used as an inducible promoter. The maximum specific (3-galactosidase activity became 58,000 Miller units, equivalent to approximately 45 % of total cellular proteins. 

The initial approach adopted entailed administration of naked plasmid DNA housing the gene of interest. This avenue of research was first opened in 1990, when it was shown that naked plasmid DNA was expressed in mice muscle cells subsequent to its i.m. injection. The plasmid DNA concerned housed the /1-galactosidase gene as a reporter. Subsequent expression of S-galactosidase activity could persist for anything from a few months to the remainder of the animal s life. The transfection rate recorded was low (1-2% of muscle fibres assimilated the DNA) and the DNA was not integrated into the host cell s chromosomes. 

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