Difference between revisions of "Part:BBa K4593003"
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<partinfo>BBa_K4593003 short</partinfo> | <partinfo>BBa_K4593003 short</partinfo> | ||
| − | + | This part is the coding sequence of endolysin GH15 | |
| − | |||
===Usage and Biology=== | ===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 unexpectedly reveals an "EF-hand-like" calcium-binding site adjacent to its active site groove, and this calcium ion modulates the domain's lytic activity; Amidase-2 domain, where both E282 and a zinc ion are integral for its lytic function; The SH3b domain, which is vital for substrate interactions[2]. | |
| − | + | ||
| − | + | ||
| + | ===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 we've tested due to its powerful bactericidal properties. We're employing it in our lysing experiments. | |
| − | === | + | |
| − | + | ====Characterization of lytic activity when expressed in E.coli==== | |
| − | + | ||
| + | To gauge the lytic efficiency of GH15, we've constructed the pET28a(+)_GH15 plasmid (assembled by Genscript) (Fig.1). This design ensures GH15 is expressed when IPTG is present. | ||
| + | |||
| + | Figure 1. The plasmid map of LysGH15+pET15b product | ||
| + | |||
| + | ====Examining protein length and purity using SDS-PAG==== | ||
| + | |||
| + | Post-purification of GH15 through nickel bead columns, its length and purity are affirmed using SDS-PAGE, which is subsequently stained with Coomassie Brilliant Blue. | ||
| + | |||
| + | Figure 2. SDS-PAGE Result of GH15 Purification | ||
| + | |||
| + | |||
| + | Endolysin LysGH15, weighing 58kDa, 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, 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, an overnight culture method was employed. After reaching an OD600 of 1.0, cells were centrifuged and resuspended in a specific reaction buffer. The first two groups were treated with water and GH15's elution buffer, while the next three received GH15 at varying concentrations. OD600 was recorded at several intervals post GH15 addition, each measured thrice. The results highlight endolysin GH15's bactericidal prowess, as a consistent decrease in OD600 values, pointing to a reduced S. aureus count, was observed. | ||
| + | |||
| + | Figure 3. OD 600 of S. aureus under different concentration of endolysin GH15 with respect to time (error bar is too small to be visible) | ||
| + | |||
| + | ====Examining lysing ability using chromogenic plates==== | ||
| + | |||
| + | For a broader view, cultures from one group were diluted and spread on S. aureus chromogenic agar 120 minutes post-induction. Overnight incubation revealed colony densities consistent with OD600 measurements. The results underline a clear relationship between the S. aureus growth rate and the lysing efficiency of E. coli, affirming the study's successful completion of this phase. | ||
| + | |||
| + | Figure 4. Overnight S.aureus chromogenic plate of S.aureus treated with different endolysin GH15 concentration for 120 min. | ||
| + | |||
| + | A) 1uM GH15 | ||
| + | |||
| + | B) elution buffer | ||
Revision as of 09:52, 27 September 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 unexpectedly reveals an "EF-hand-like" calcium-binding site adjacent to its active site groove, and this calcium ion modulates the domain's lytic activity; Amidase-2 domain, where both E282 and a zinc ion are integral for its lytic function; The SH3b domain, which is vital for substrate interactions[2].
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 we've tested due to its powerful bactericidal properties. We're employing it in our lysing experiments.
Characterization of lytic activity when expressed in E.coli
To gauge the lytic efficiency of GH15, we've constructed the pET28a(+)_GH15 plasmid (assembled by Genscript) (Fig.1). This design ensures GH15 is expressed when IPTG is present.
Figure 1. The plasmid map of LysGH15+pET15b product
Examining protein length and purity using SDS-PAG
Post-purification of GH15 through nickel bead columns, its length and purity are affirmed using SDS-PAGE, which is subsequently stained with Coomassie Brilliant Blue.
Figure 2. SDS-PAGE Result of GH15 Purification
Endolysin LysGH15, weighing 58kDa, 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, 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, an overnight culture method was employed. After reaching an OD600 of 1.0, cells were centrifuged and resuspended in a specific reaction buffer. The first two groups were treated with water and GH15's elution buffer, while the next three received GH15 at varying concentrations. OD600 was recorded at several intervals post GH15 addition, each measured thrice. The results highlight endolysin GH15's bactericidal prowess, as a consistent decrease in OD600 values, pointing to a reduced S. aureus count, was observed.
Figure 3. OD 600 of S. aureus under different concentration of endolysin GH15 with respect to time (error bar is too small to be visible)
Examining lysing ability using chromogenic plates
For a broader view, cultures from one group were diluted and spread on S. aureus chromogenic agar 120 minutes post-induction. Overnight incubation revealed colony densities consistent with OD600 measurements. The results underline a clear relationship between the S. aureus growth rate and the lysing efficiency of E. coli, affirming the study's successful completion of this phase.
Figure 4. Overnight S.aureus chromogenic plate of S.aureus treated with different endolysin GH15 concentration for 120 min.
A) 1uM GH15
B) elution buffer