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
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This part is the coding sequence of endolysin LysGH15
  
 
===Usage and Biology===
 
===Usage and Biology===
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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 we've tested due to its powerful bactericidal properties. We're employing it in our lysing experiments.
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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 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.
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To get purified LysGH15, we've designed the pET15b(+)_GH15 plasmid (assembled by Genscript) (Fig.1). This design allows LysGH15 to be expressed when IPTG is present.
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<html>
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<figure>
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/4593/wiki/result/gh15-map.png" width="700" height="auto"/>
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</figcaption>
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</figure>
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Figure 1. The plasmid map of LysGH15+pET15b product
 
Figure 1. The plasmid map of LysGH15+pET15b product
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====Examining protein length and purity using SDS-PAG====
 
====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.
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Purification of LysGH15 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
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<html>
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<figure>
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/4593/wiki/result/jiaotu/gh15.png" width="400" height="auto"/>
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</figcaption>
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</figure>
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</html>
  
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Figure 2. SDS-PAGE Result of LysGH15 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.
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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 LysGH15 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 LysGH15, 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, 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.
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To evaluate endolysin LysGH15'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 LysGH15, respectively, while the subsequent three were treated with LysGH15 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 LysGH15 addition, with each time point being repeated three times.
  
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)
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<html>
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<figure>
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/4593/wiki/result/gh15.png" width="700" height="auto"/>
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</figcaption>
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</figure>
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</html>
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Figure 3. OD 600 of S. aureus under different concentrations of endolysin LysGH15 with respect to time (error bars are too small to be visible)
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The data emphasizes the bactericidal activity of endolysin LysGH15. 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====
 
====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.
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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 concentration for 120 min.  
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<html>
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<figure>
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/4593/wiki/result/gh15-pic.jpg" width="700" height="auto"/>
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</figcaption>
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</figure>
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</html>
  
A) 1uM GH15
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Figure 4. Overnight S.aureus chromogenic plate of S.aureus treated with different endolysin LysGH15 concentrations for 120 min.
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A) 1uM LysGH15
  
 
B) elution buffer
 
B) elution buffer
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4593003 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K4593003 parameters</partinfo>
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Latest revision as of 18:31, 3 October 2023


LysGH15

This part is the coding sequence of endolysin LysGH15

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 LysGH15, we've designed the pET15b(+)_GH15 plasmid (assembled by Genscript) (Fig.1). This design allows LysGH15 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 LysGH15 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 LysGH15 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 LysGH15 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 LysGH15, even if yields are lower.

Examining lysing ability using spectrophotometer

To evaluate endolysin LysGH15'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 LysGH15, respectively, while the subsequent three were treated with LysGH15 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 LysGH15 addition, with each time point being repeated three times.

Figure 3. OD 600 of S. aureus under different concentrations of endolysin LysGH15 with respect to time (error bars are too small to be visible)

The data emphasizes the bactericidal activity of endolysin LysGH15. 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 LysGH15 concentrations for 120 min.

A) 1uM LysGH15

B) elution buffer


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 165
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 649
    Illegal NgoMIV site found at 1323
    Illegal AgeI site found at 793
    Illegal AgeI site found at 1021
    Illegal AgeI site found at 1192
    Illegal AgeI site found at 1305
  • 1000
    COMPATIBLE WITH RFC[1000]