Composite

Part:BBa_K1659301

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

Micrococcal Nuclease (DNase) fused at N-terminal with DsbA signal sequence


This part contains the sequence for the Staphylococcus aureus-derived enzyme, micrococcal DNase (also known as staphylococcal nuclease or thermonuclease) fused with an export signal at its N-terminus.


Biology

BBa_K1659301 is a composite of Micrococcal DNase (BBa_K1659300) with the 2-19 peptide segment of protein-folding factor DsbA:


1. Micrococcal DNase

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].


2. DsbA 2-19 signal sequence

DsbA is a thioredoxin fold-containing disulfide oxidoreductase protein found predominantly in Gram-negative bacteria, which functions as a protein-folding factor [12][13]. The 2-19 peptide sequence of DsbA is a signal sequence that can direct passenger proteins for co-translational export via the signal recognition particle (SRP) pathway [14][15]. It has recently been shown that the DsbA signal sequence is capable of mediating passenger protein secretion under a selection of different induction temperatures [16].


Usage

We fused the DsbA 2-19 signal peptide sequence to the N-terminus of micrococcal DNase to with the aim of facilitating the fusion protein's export via the SRP pathway. A hexahistidine tag is also attached onto the C-terminus of the composite to allow for easy purification of the expressed protein via metal-affinity column chromatography.

In terms of scaling up the recombinant enzyme prooduction, it would be more desirable and efficient for the enzyme product to be available extracellularly as a secreted product rather than intracellularly, as the former would allow for a more streamlined harvesting process involving only the collection of the secretant-containing extracellular media as opposed to the need to process the host cells for batch lysis during each harvest.

As far as enzyme function is concerned, 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. Ultimately, we aim to use antibiofilm enzymes such as micrococcal DNase in conjunction with antibacterial enzymes such as Art-175 as an alternative treatment option to antibiotics in biofilm-related bacterial infections.

To characterize this part, we moved the DsbA-DNase coding sequence into the commercial expression vector pBAD/HisB by adding a BspHI restriction site to the 5' site of the coding sequence using PCR and performing digestion-ligation at BspHI(insert)-NcoI(plasmid) and PstI, making the expression of the DsbA-DNase coding gene inducible by L-arabinose. This DsbA-DNase[pBAD] plasmid is then cloned into E. coli MG1655.

Purification of Secreted Protein

MG1655 DsbA-DNase[pBAD] subcultured 1:20 in LB media (total volume 500mL), grown at 37°C for 1 hour then induced with 0.2% arabinose at 30°C for 4 hours. Supernatant purified using nickel-affinity chromatography using the following set of buffers:

- Resuspension buffer: 50 mM sodium phosphate, pH 8.0, 0.3 M sodium chloridine. - Wash buffer: 50 mM sodium phosphate, pH 8.0, 0.3 M sodium chloridine and 10mM imidazole. - Elution buffer: 50 mM sodium phosphate, pH 8.0, 0.3 M sodium chloridine and 250mM imidazole.

10uL of eluate was mixed with SDS and run through a PAGE gel:

Lane C is where the stained eluate was loaded (DsbA-DNase is a 21kDa protein); Ladder used was 2-Color SDS Marker

This shows DsbA signal sequence successfully facilitates the export of DNase from MG1655.

Inhibition of Host Cell Biofilm Formation

Stationary cultures of and MG1655 DsbA-DNase[pBAD] subcultured 1:100 in fresh LB media and inoculated into 96-well plate incubated at room temperature for 3 days with or without 0.2% arabinose as gene expression inducer accordingly. Planktonic cells removed through very gentle rinsing with Milli-Q water, and adherent biofilms stained using 0.1% crystal violet solution. Stained biofilms dissolved in 80-20 ethanol-acetone and optical density at 590 nm measured (the higher the OD, the more stained biofilm).

Analysis was done alongside BBa_K1659211


Data shows that expression of DsbA-DNase inhibits host cell biofilm formation.


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.

[12] Guddat, L.W., Bardwell, J.C. & Martin, J.L., 1998. Crystal structures of reduced and oxidized DsbA: investigation of domain motion and thiolate stabilization. Structure (London, England : 1993), 6(6), pp.757–767.

[13] Heras, B. et al., 2009. DSB proteins and bacterial pathogenicity. Nature reviews. Microbiology, 7(3), pp.215–225.

[14] Schierle, C.F. et al., 2003. The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway. Journal of Bacteriology, 185(19), pp.5706–5713.

[15] Steiner, D. et al., 2006. Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. Nature biotechnology, 24(7), pp.823–831.

[16] Božić, N. et al., 2013. The DsbA signal peptide-mediated secretion of a highly efficient raw-starch-digesting, recombinant α-amylase from Bacillus licheniformis ATCC 9945a. Process Biochemistry, 48(3), pp.438–442.

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