Difference between revisions of "Part:BBa K4593000"

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To get purified LysDZ25, we've designed the pET28a(+)_DZ25 plasmid (assembled by Genscript) (Fig.1). This design allows LysDZ25 to be expressed when IPTG is present.
 
To get purified LysDZ25, we've designed the pET28a(+)_DZ25 plasmid (assembled by Genscript) (Fig.1). This design allows LysDZ25 to be expressed when IPTG is present.
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/4593/wiki/result/dz25-map.png" width="700" height="auto"/>
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Figure 1. The plasmid map of pET28a(+)_DZ25
 
Figure 1. The plasmid map of pET28a(+)_DZ25
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Purification of LysDZ25 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.
 
Purification of LysDZ25 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.
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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/dz25.png" width="240" height="auto"/>
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</figcaption>
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</figure>
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</html>
  
 
Figure 2. SDS-PAGE Result of LysDZ25 Purification
 
Figure 2. SDS-PAGE Result of LysDZ25 Purification
  
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Endolysin LysDZ25, possessing a molecular weight of 58kDa, presented a distinct band in lane 2, closely aligning with the 55kDa marker on the ladder, pointing towards the successful purification of DZ25. However, it appears that the final wash did not completely remove all contaminant proteins, potentially introducing some degree of uncertainty to the findings.
+
Endolysin LysDZ25, possessing a molecular weight of 58kDa, presented a distinct band in lane 2, closely aligning with the 55kDa marker on the ladder, pointing towards the successful purification of LysDZ25. However, it appears that the final wash did not completely remove all contaminant proteins, potentially introducing some degree of uncertainty to the findings.
  
−
The purity of DZ25, as evident from its lane on the gel, was lower than other proteins used in our project, implying the presence of approximately 50% other proteins in the mixture. Nonetheless, this deviation did not impede the subsequent applications of DZ25, which still exhibited a promising capability in sterilizing S. aureus.
+
The purity of LysDZ25, as evident from its lane on the gel, was lower than other proteins used in our project, implying the presence of approximately 50% other proteins in the mixture. Nonetheless, this deviation did not impede the subsequent applications of LysDZ25, which still exhibited a promising capability in sterilizing S. aureus.
  
 
====Examining lysing ability using spectrophotometer====
 
====Examining lysing ability using spectrophotometer====
  
−
The bactericidal activity of endolysin DZ25 against S. aureus was evaluated under various concentrations. An overnight culture of S. aureus was diluted 500-fold into fresh TSB medium. The cells were then centrifuged once the OD600 reached a value of 2.0. The pelleted cells were then re-suspended in a reaction buffer (20 mM Tris, 300 mM NaCl, pH 8.0). 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.
+
The bactericidal activity of endolysin LysDZ25 against S. aureus was evaluated under various concentrations. An overnight culture of S. aureus was diluted 500-fold into fresh TSB medium. The cells were then centrifuged once the OD600 reached a value of 2.0. The pelleted cells were then re-suspended in a reaction buffer (20 mM Tris, 300 mM NaCl, pH 8.0). The initial two groups received 1ml of water and the elution buffer of LysDZ25, respectively, while the subsequent two were treated with LysDZ25 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 LysDZ25 addition, with each time point being repeated three times.
  
−
The data underscore the formidable bactericidal properties of endolysin DZ25. As time progressed, we observed a marked decline in the OD600 values, indicating a reduction in the S. aureus colony density. Also, the rate of bacterial elimination was faster at a concentration of 0.01mg/ml compared to 0.05mg/ml, especially in the initial 5 and 20-minute intervals. This unexpected result could not be explained - the salt concentration difference between the two groups should be the same, and even though there is a minor difference, it should not affect the activity of DZ25 due to its wide optimal salt concentration [2]. However, by the one-hour mark, both concentrations had effectively reduced the bacterial count, exhibiting only minor differences in their overall efficacy.
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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/dz25.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 LysDZ25 with respect to time (some error bars are too small to be visible)
  
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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)
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The data underscore the formidable bactericidal properties of endolysin LysDZ25. As time progressed, we observed a marked decline in the OD600 values, indicating a reduction in the S. aureus colony density. Also, the rate of bacterial elimination was faster at a concentration of 0.01mg/ml compared to 0.05mg/ml, especially in the initial 5 and 20-minute intervals. This unexpected result could not be explained - the salt concentration difference between the two groups should be the same, and even though there is a minor difference, it should not affect the activity of LysDZ25 due to its wide optimal salt concentration [2]. However, by the one-hour mark, both concentrations had effectively reduced the bacterial count, exhibiting only minor differences in their overall efficacy.
  
 
====Examining lysing ability using chromogenic plates====
 
====Examining lysing ability using chromogenic plates====
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To offer a more comprehensive perspective, cultures from each group were diluted 500-fold and plated on S. aureus chromogenic agar post 120 minutes of reaction. 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.
 
To offer a more comprehensive perspective, cultures from each group were diluted 500-fold and plated on S. aureus chromogenic agar post 120 minutes of reaction. 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.
  
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<figure>
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/4593/wiki/result/dz25-pic.jpg" width="700" height="auto"/>
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</figcaption>
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</figure>
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Figure 4. Overnight S.aureus chromogenic plate of S.aureus treated with different endolysin LysDZ25 concentrations for 120 min.
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A) 0.05mg/ml LysDZ25     
  
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Figure 4. Overnight S.aureus chromogenic plate of S.aureus treated with different endolysin GH15 concentrations for 120 min.  
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B) 0.01mg/ml LysDZ25   
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C) water       
  
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A) 1uM GH15
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D) Elution buffer
  
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B) elution buffer
 
  
  

Latest revision as of 18:30, 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 LysDZ25 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 LysDZ25 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 LysDZ25 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 LysDZ25 Purification

Endolysin LysDZ25, possessing a molecular weight of 58kDa, presented a distinct band in lane 2, closely aligning with the 55kDa marker on the ladder, pointing towards the successful purification of LysDZ25. However, it appears that the final wash did not completely remove all contaminant proteins, potentially introducing some degree of uncertainty to the findings.

The purity of LysDZ25, as evident from its lane on the gel, was lower than other proteins used in our project, implying the presence of approximately 50% other proteins in the mixture. Nonetheless, this deviation did not impede the subsequent applications of LysDZ25, which still exhibited a promising capability in sterilizing S. aureus.

Examining lysing ability using spectrophotometer

The bactericidal activity of endolysin LysDZ25 against S. aureus was evaluated under various concentrations. An overnight culture of S. aureus was diluted 500-fold into fresh TSB medium. The cells were then centrifuged once the OD600 reached a value of 2.0. The pelleted cells were then re-suspended in a reaction buffer (20 mM Tris, 300 mM NaCl, pH 8.0). The initial two groups received 1ml of water and the elution buffer of LysDZ25, respectively, while the subsequent two were treated with LysDZ25 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 LysDZ25 addition, with each time point being repeated three times.

Figure 3. OD 600 of S. aureus under different concentrations of endolysin LysDZ25 with respect to time (some error bars are too small to be visible)

The data underscore the formidable bactericidal properties of endolysin LysDZ25. As time progressed, we observed a marked decline in the OD600 values, indicating a reduction in the S. aureus colony density. Also, the rate of bacterial elimination was faster at a concentration of 0.01mg/ml compared to 0.05mg/ml, especially in the initial 5 and 20-minute intervals. This unexpected result could not be explained - the salt concentration difference between the two groups should be the same, and even though there is a minor difference, it should not affect the activity of LysDZ25 due to its wide optimal salt concentration [2]. However, by the one-hour mark, both concentrations had effectively reduced the bacterial count, exhibiting only minor differences in their overall efficacy.

Examining lysing ability using chromogenic plates

To offer a more comprehensive perspective, cultures from each group were diluted 500-fold and plated on S. aureus chromogenic agar post 120 minutes of reaction. 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 LysDZ25 concentrations for 120 min.

A) 0.05mg/ml LysDZ25

B) 0.01mg/ml LysDZ25

C) water

D) Elution buffer


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]