Difference between revisions of "Part:BBa K4593003"

Revision as of 17:48, 3 October 2023

LysGH15

This part is the coding sequence of endolysin GH15

Usage and Biology

Derived from the staphylococcal phage GH15, Endolysin LysGH15 demonstrates a robust ability to lyse Staphylococcus aureus. LysGH15 comprises three domains: The CHAP domain, which is a calcium-dependent Enzyme Active Domain (EAD); the Amidase-2 domain, an EAD that both E282 and a zinc ion are integral for its lytic function; and the SH3b domain, a Cell-Binding Domain (CBD) is vital for substrate interactions​[1]​.

Team: BNDS-China 2023

Our project aims to create a suite of effective methods for both detecting and lysing S. aureus. Within this framework, LysGH15 stands out as one of the endolysins with powerful bactericidal properties. We're employing it in our lysing experiments.

Characterization of lytic activity when expressed in E.coli

To get purified GH15, we've designed the pET15b(+)_GH15 plasmid (assembled by Genscript) (Fig.1). This design allows GH15 to be expressed when IPTG is present.

Figure 1. The plasmid map of LysGH15+pET15b product

Examining protein length and purity using SDS-PAG

Purification of GH15 is done through nickel bead columns. Its length and purity are affirmed using SDS-PAGE, which is subsequently stained with Coomassie Brilliant Blue for visualization.

Figure 2. SDS-PAGE Result of GH15 Purification

Endolysin LysGH15, weighing approximately 58kDa (with tags), showed a prominent band close to the 55kDa ladder marker in lanes 3, 4, and 5, confirming the GH15 protein's expected length. The clear lane 2 indicates sufficient washing under the current washing concentration (20mM imidazole), but to increase protein concentration in the eluate, a higher imidazole concentration than the current 20mM might be needed. Despite this, lane 4 displays a high purity of GH15, even if yields are lower.

Examining lysing ability using spectrophotometer

To evaluate endolysin GH15's capacity to neutralize S. aureus, S. aureus is cultured overnight and then diluted in a fresh TSB medium. After reaching an OD600 of 1.0, cells were centrifuged, resuspended by reaction buffer (20 mM PBS, 500 mM NaCl, pH 8.0), and divided into 4 groups with identical initial OD600 (about 1.0). The initial two groups received 1ml of water and the elution buffer of GH15, respectively, while the subsequent three were treated with GH15 to achieve concentrations (0.25uM, 0.05uM, and 1uM). The OD600 for each group was recorded just after the endolysin was added, and at intervals of 5, 10, 15, 20, 30, and 60 minutes post GH15 addition, with each time point being repeated three times.

Figure 3. OD 600 of S. aureus under different concentrations of endolysin GH15 with respect to time (error bars are too small to be visible)

The data emphasizes the bactericidal activity of endolysin GH15. As the experiment progressed, a significant decline in the OD600 measurements became evident, indicative of a diminishing S. aureus cell density. Notably, the rate of bacterial elimination was highest at the 1uM concentration, while efficiency reduced at 0.25uM and 0.05uM concentrations, especially during the initial 0-20 minutes. By the end of the one-hour period, both the 1uM and 0.25uM concentrations had markedly reduced bacterial counts, showing only small differences in their overall effectiveness. However, the 0.05uM concentration lagged considerably behind, displaying a lack of bactericidal capability.

Examining lysing ability using chromogenic plates

To offer a more comprehensive perspective, cultures from the 1uM and elusion buffer group were diluted 300-fold and spread on S. aureus chromogenic agar post 120 minutes of reaction (the rest two groups were not joined because there weren’t enough chromogenic plates). The colonies' density after an overnight incubation corroborated the insights derived from the OD600 assessments. Altogether, the findings confirm the successful execution of this part of the study.

Figure 4. Overnight S.aureus chromogenic plate of S.aureus treated with different endolysin GH15 concentrations for 120 min.

A) 1uM GH15

B) elution buffer

Sequence and Features