Difference between revisions of "Part:BBa K2609001"

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<h2> Biology </h2>
 
<h2> Biology </h2>
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<p>The T4 Bacteriophage uses the holin-endolysin system for host lysis. The bacteriophage resorts to active degradation of petidoglycan with endolysin. Endolysin, which has no secretory signal sequence, accumulates in a fully folded and active state in the cytosol but is incapable of unassisted escape from the cytoplasm. This requires a pathway for the endolysin to reach the cell wall for it to act on peptidoglycan and it is holin that triggers and controls the host cell wall degradation at the end of lytic cycle. Holin creates pores in the inner membrane allowing the lysozyme to reach the peptidoglycan region from the cytosol. Here hole is refers to "Holin mediated permeabilizing lesion". This basically is associated with a collapse of the membrane potential and thus permeabilizing the membrane.<sup>[1][2]</sup></p>  
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<p>The T4 Bacteriophage uses the holin-endolysin system for host lysis. The bacteriophage resorts to active degradation of petidoglycan with endolysin. Endolysin, which has no secretory signal sequence, accumulates in a fully folded and active state in the cytosol but is incapable of unassisted escape from the cytoplasm. This requires a pathway for the endolysin to reach the cell wall for it to act on peptidoglycan and it is holin that triggers and controls the host cell wall degradation at the end of lytic cycle. Holin creates pores in the inner membrane allowing the lysozyme to reach the peptidoglycan region from the cytosol. Here, hole refers to "Holin mediated permeabilizing lesion". This basically is associated with a collapse of the membrane potential and thus permeabilizing the membrane.<sup>[1][2]</sup></p>  
  
 
<h2>Usage</h2>
 
<h2>Usage</h2>
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<h3>IISc Bangalore 2018</h3>
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The 2018 IISc-Bangalore iGEM team used this sequence with the constitutive promoter (BBa_J23106) to make <a href="https://parts.igem.org/Part:BBa_K2609017">BBa_K2609017</a> for screening of recombinants after Lambda Red recombination.</p>
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<p>We used the sequence with the constitutive promoter (BBa_J23106) to make <a href="https://parts.igem.org/Part:BBa_K2609017">BBa_K2609017</a> for screening of recombinants after Lambda Red recombination.</p>
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<center><img src="https://static.igem.org/mediawiki/parts/6/6d/T--IISc-Bangalore--endolysinreplica.png" width=80% style="border: 1px solid black;"></center>
  
 
<h2>Characterization</h2>
 
<h2>Characterization</h2>
 
<h3>IISc Bangalore 2018</h3>
 
<h3>IISc Bangalore 2018</h3>
  
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<h4>Expression and characterization</h4>
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<h4>Expression and characterization with <a href="https://parts.igem.org/Part:BBa_K2609017">BBa_K2609017</a></h4>
 
<p>The part was transformed into <I>E.coli</I> BL21(DE3). We ran an SDS-PAGE of the cell lysate of the transformed clones against the protein ladder.The protein has a size~18.7 kDa. However due to the presence of <I>E.coli</I> proteins of similar size, there is no significant difference seen in the two lanes<sup>[3]</sup>. We characterized the protein by checking for its lytic activity as mentioned below.</p>
 
<p>The part was transformed into <I>E.coli</I> BL21(DE3). We ran an SDS-PAGE of the cell lysate of the transformed clones against the protein ladder.The protein has a size~18.7 kDa. However due to the presence of <I>E.coli</I> proteins of similar size, there is no significant difference seen in the two lanes<sup>[3]</sup>. We characterized the protein by checking for its lytic activity as mentioned below.</p>
 
<center><img src="https://static.igem.org/mediawiki/parts/b/b6/T4_Endolysin_under_constitutive_promoter_.png" width=30% style="border: 1px solid black;"></center>
 
<center><img src="https://static.igem.org/mediawiki/parts/b/b6/T4_Endolysin_under_constitutive_promoter_.png" width=30% style="border: 1px solid black;"></center>

Latest revision as of 00:13, 18 October 2018

T4 endolysin

The Endolysin(e) gene from the T4 bacteriophage. Works as a lysozyme and degrades the peptidoglycan cell wall at the end of the lytic cycle of bacteriophage infection. Extracellularly bactericidal for gram-positive bacteria. Gram negative bacteria are resistant to lysis induced by external endolysin because of the presence of an outer memnrane.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 255
    Illegal AgeI site found at 325
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Biology

The T4 Bacteriophage uses the holin-endolysin system for host lysis. The bacteriophage resorts to active degradation of petidoglycan with endolysin. Endolysin, which has no secretory signal sequence, accumulates in a fully folded and active state in the cytosol but is incapable of unassisted escape from the cytoplasm. This requires a pathway for the endolysin to reach the cell wall for it to act on peptidoglycan and it is holin that triggers and controls the host cell wall degradation at the end of lytic cycle. Holin creates pores in the inner membrane allowing the lysozyme to reach the peptidoglycan region from the cytosol. Here, hole refers to "Holin mediated permeabilizing lesion". This basically is associated with a collapse of the membrane potential and thus permeabilizing the membrane.[1][2]

Usage

The 2018 IISc-Bangalore iGEM team used this sequence with the constitutive promoter (BBa_J23106) to make BBa_K2609017 for screening of recombinants after Lambda Red recombination.

Characterization

IISc Bangalore 2018

Expression and characterization with BBa_K2609017

The part was transformed into E.coli BL21(DE3). We ran an SDS-PAGE of the cell lysate of the transformed clones against the protein ladder.The protein has a size~18.7 kDa. However due to the presence of E.coli proteins of similar size, there is no significant difference seen in the two lanes[3]. We characterized the protein by checking for its lytic activity as mentioned below.

Characterisation of lytic activity of Endolysin

The lytic activity of endolysin was characterised by measuring the decrease in the optical density of B.cereus (gram positive) cell suspension after addition of endolysin[4][5]. Exponentially growing Bacillus cereus cells were washed twice and resuspended in 0.2mM Tris-HCl(pH-8) to adjust to OD600nm= 0.6-0.8. Then Endolysin protein(100 µL) was added to 900µL of cell suspension. The following were used as controls:
1. 900µL cell suspension with 100µL of resuspension buffer.
2. 900µL cell suspension with 100µL of Lysis Buffer used in protein extraction.
3. 900µL cell suspension with 100µL of Proteins from Wild Type E.coli(BL21(DE3))
The OD600nm at 37o was measured using a plate reader at regular intervals of time and the lytic activity was noted from the Change in OD v/s time curve as in Figure 2.

Endolysin expressed from BBa_K2609017 has a much stronger lytic activity compared to the buffer or normal cell lysate controls. Hence, the part works as expected and is extracellulary disruptive to gram positive bacteria.

References

[1]Eric S. Miller, Elizabeth Kutter,et al."Bacteriophage T4 Genome". Microbiol. Mol. Biol. Rev. Mar 2003, 67 (1) 86-156; DOI: 10.1128/MMBR.67.1.86-156.2003
[2]Wang, I N et al. “Holins: the protein clocks of bacteriophage infections.” Annual review of microbiology 54 (2000): 799-825.
[3]Lim, Jeong-A YangᆞSoon-Ryun et al. “Exogenous lytic activity of SPN9CC endolysin against gram-negative bacteria.” Journal of microbiology and biotechnology 24 6 (2014): 803-11.
[4]Jaeeun Park, Jiae Yun, et al. "Characterization of an endolysin, LysBPS13, from a Bacillus cereus bacteriophage", FEMS Microbiology Letters, Volume 332, Issue 1, 1 July 2012, Pages 76–83, https://doi.org/10.1111/j.1574-6968.2012.02578.x
[5]Lim, Jeong-A, et al. "Characterization of Endolysin From a Salmonella Typhimurium-infecting Bacteriophage Spn1s." Research in microbiology 163.3 (2012): 233-241. doi: 10.1016/j.resmic.2012.01.002