Difference between revisions of "Part:BBa K4593000"

Revision as of 17:37, 3 October 2023

Endolysin LysDZ25

This part is the coding sequence of endolysin LysDZ25

Usage and Biology

Derived from the staphylococcal phage DZ25 (originally isolated from milk), Endolysin DZ25 demonstrates a robust ability to lyse Staphylococcus aureus. LysDZ25 comprises three domains: two Enzymatically Active Domains (EDS) and a Cell-Binding Domain (CBD). However, the location of these domains are not specified in the article [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, LysDZ25 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 LysDZ25, we've designed the pET28a(+)_DZ25 plasmid (assembled by Genscript) (Fig.1). This design allows DZ25 to be expressed when IPTG is present.

Figure 1. The plasmid map of pET28a(+)_DZ25

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 Tris, 300 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 DZ25, respectively, while the subsequent two were treated with DZ25 to achieve concentrations of 0.05mg/mL and 0.01mg/mL. 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 after the DZ25 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)

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