Difference between revisions of "Part:BBa K4593003"
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Jianfei Song (Talk | contribs) (→Team: BNDS-China 2023) |
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===Team: BNDS-China 2023=== | ===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 | + | 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==== | ====Characterization of lytic activity when expressed in E.coli==== | ||
| − | To | + | To get purified GH15, we've designed the pET15b(+)_GH15 plasmid (assembled by Genscript) (Fig.1). This design allows GH25 to be expressed when IPTG is present. |
Figure 1. The plasmid map of LysGH15+pET15b product | Figure 1. The plasmid map of LysGH15+pET15b product | ||
| Line 21: | Line 21: | ||
====Examining protein length and purity using SDS-PAG==== | ====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 | 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. | + | 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==== | ====Examining lysing ability using spectrophotometer==== | ||
| − | To evaluate endolysin GH15's capacity to neutralize S. aureus, | + | 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 | + | 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==== | ====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 | + | Figure 4. Overnight S.aureus chromogenic plate of S.aureus treated with different endolysin GH15 concentrations for 120 min. |
A) 1uM GH15 | A) 1uM GH15 | ||
B) elution buffer | B) elution buffer | ||
Revision as of 15:02, 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 GH25 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 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