Development of an LacZ Based Arsenic Biosensor [344

Understanding the mechanism for arsenic resistance is necessary in order to develop an appropriate biosensor and to better understand its response. Therefore, to develop the biosensor, the arsenic-resistant gene and the reporter gene must be cloned and inserted onto one plasmid, which then is inserted into a host bacterium. The arsenic-sensing biosensor will then be triggered by arsenic, the analyte, entering the biosensor and activating the transcription of the resistant gene, which is to be followed by the transcription of the reporter gene. The entire resistant gene may not be needed, and the biosensor can only use the beginning components, such as the promoter, and should be able to recognize the arsenic and begin the transcription of the plasmid that contains the reporter gene.

The transcription of the reporter gene will produce proteins whose activity will be assessed in direct correlation to the amount of arsenic entering it. Bacterial arsenic resistance genetic operon arsRDABC or arsRBC present in diverse bacterial species have been well characterized [24,136]. Because the ars operator-promoter is inducible by arsenite (or arsenate in the presence of arsC),and inducibility is positively correlated with the concentration of the inducer, the activity of a reporter gene product, if cloned under this promoter, will reflect the availability of the inducer/analyte.

In the present study, an arsenic biosensor was created using the lacZ reporter gene. The promoterless lacZ gene, lacking a ribosome-binding sequence and the first eight nonessential amino acid codons, was coupled with the ars promoter along with arsR, arsD, and a part of ars A gene in the translational lacZ fusion vector, pMC1871. The recombinant plasmid pASH3, having truncated the ars operon, was found hypersensitive to arsenite and arsenate and the phenomenon was found suitable to develop a simple bioassay system for arsenic. However, it is not known whether the hypersensitivity to arsenite rendered by pASH3 is associated with active arsenite uptake like the mercury-hypersensitive clone. Thus far, the hypersensitivity of arsenite observed in E. coli cells conferred by pASH3 cannot be explained from the present state of knowledge of arsenic resistance and or uptake in microorganisms. However, the expression of ars-lacZ fusion was expected at a concentration lower than the values observed earlier with the complete ars operon arsRDABC.

The maximum specific activity was obtained with arsenite between 0.5 and 1 |M; however, 0-galactosidase activity was found inducible even with 10 nM of arsenite. Therefore, the clone pASH3 could serve a biosensor to detect a very low concentration of arsenic, which may not be easily detectable by the standard chemical methods. When the arsenite-responsive regulation unit of the plasmid pI258 was used to express the firefly luciferase gene (lucFF), the lowest arsenite concentration required to induce the reporter gene was 100 nM and maximum induction was noted at 3.3 mM4 4 arsenite. In another method, expression of arsD-lacZ fusion was monitored by an electro chemical reaction to assay the arsenite-dependent P-galactosidase activity. The detection limit was 100 nM, but a 17-h induction period was required.

These two methods are apparently suitable for environmental samples of low bioavailable arsenic content; however, both require relatively costly instruments and may not be suitable in a field study. There are few reports of successful gene-fusion biosensors in the monitoring of a metal toxicity in field application. For instance, a luminescent bacterial biosensor was shown to be effective in the evaluation of arsenic bioavailability of chromated copper arsenate contamination [343]. Detection limit of arsenite by arsenic hypersensitive clone pASH3 was comparable with the earlier claims; the methodology is relatively simple as well. Moreover, the process can easily be improvised to an acceptable arsenic assay kit with a low-cost investment for monitoring a large number of samples for on-site analysis.

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