Coding

Part:BBa_K1659300

Designed by: Wei Chung Kong   Group: iGEM15_Oxford15   (2015-08-28)

Micrococcal Nuclease (DNase)


This part contains the sequence for the Staphylococcus aureus-derived enzyme, micrococcal DNase (also known as staphylococcal nuclease or thermonuclease).


Biology

Micrococcal DNase is an endo-exonuclease that non-specifically catalyzes the hydrolysis of single- and double-stranded DNA under basic conditions and in the presence of Ca2+ ions, and is known to be able to speed up DNA hydrolysis by up to 1016 times [4].

The crystal structure of micrococcal DNase was resolved in 1971, long before the endogeneous function of said enzyme was discovered [1]. As the enzyme's relatively simple structure proved to be very helpful towards the study of its biochemical and physical studies, researchers rapidly went on to identify the gene responsible for its synthesis and clone said gene in different expression hosts for heterologous characterization [2][3][5]. Micrococcal DNase test agars are also a well-known indicator of S. aureus contamination [6].

It is a well-established fact that extracellular DNA is a vital structural component of bacterial biofilms, with aerosols of human recombinant DNase I having been employed as a remedy for P. aeruginosa biofilms in cystic fibrosis for two decades now [7][8][9]. Recently, micrococcal DNase has been shown to be able to inhibit the formation of bacterial biofilms [10][11].


Usage

We are interested in the antibiofilm activity of micrococcal DNase against the biofilms formed by antibiotic-resistant strains of E. coli and P. aeruginosa found in urinary tract infections. However, in the interest of lab usage safety, for our wet lab work we will only test the antibiofilm potency of micrococcal DNase against Biosafety Level 1 laboratory strains of E. coli and P. putida.


References

[1] Arnone, A. et al., 1971. A High Resolution Structure of an Inhibitor the Extracellular Nuclease of Staphylococcus Complex aureus of. Journal of Biological Chemistry, 246(7), pp.2302–2316.

[2] Shortle, D., 1983. A genetic system for analysis of staphylococcal nuclease. Gene, 22(2-3), pp.181–189.

[3] Miller, J.R., Kovacevic, S. & Veal, L.E., 1987. Secretion and processing of staphylococcal nuclease by Bacillus subtilis. Journal of Bacteriology, 169(8), pp.3508–3514.

[4] Hale, S.P., Poole, L.B. & Gerlt, J. a, 1993. Mechanism of the reaction catalyzed by staphylococcal nuclease: identification of the rate-determining step. Biochemistry, 32(29), pp.7479–7487.

[5] Trémillon, N. et al., 2010. Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor. Microbial cell factories, 9, p.37.

[6] Ratner, H.B. & Stratton, C.W., 1985. Thermonuclease test for same-day identification of Staphylococcus aureus in blood cultures. Journal of Clinical Microbiology, 21(6), pp.995–996.

[7] Montanaro, L. et al., 2011. Extracellular DNA in biofilms. International Journal of Artificial Organs, 34(9), pp.824–831.

[8] Okshevsky, M. & Meyer, R.L., 2013. The role of extracellular DNA in the establishment, maintenance and perpetuation of bacterial biofilms. Critical reviews in microbiology, 7828(September), pp.1–11. Available at: http://www.ncbi.nlm.nih.gov/pubmed/24303798.

[9] Rubin, B.K., 1992. Aerosolized recombinant human deoxyribonuclease I in the treatment of cystic fibrosis. The New England journal of medicine, 327(8), p.571.

[10] Mann, E.E. et al., 2009. Modulation of eDNA release and degradation affects Staphylococcus aureus biofilm maturation. PLoS ONE, 4(6).

[11] Tang, J.N. et al., 2011. The staphylococcal nuclease prevents biofilm formation in Staphylococcus aureus and other biofilm-forming bacteria. Science China Life Sciences, 54(9), pp.863–869.

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