Difference between revisions of "Part:BBa K2609017"
 
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<partinfo>BBa_K2609017 short</partinfo>
 
<partinfo>BBa_K2609017 short</partinfo>
  
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Expresses the T4 endolysion gene under BBa_J23106, a medium strength constitutive promoter. This part was used to screen for recombined phages after Lambda Red recombination.
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This generator expresses the T4 endolysin protein (a phage lysozyme) under BBa_J23106, a medium strength constitutive promoter. Endolysin acts against the peptidoglycan layer and is minimally toxic to bacterial cells when produced internally because of the presence of an internal lipid membrane. An exposure of the peptidoglycan layer to endolysin however results in its disruption. Gram negative bacteria are resistant to lysis induced by external endolysin because of the presence of an outer membrane.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2609017 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_K2609002 parameters</partinfo>
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===Usage and Biology===
 
===Usage and Biology===
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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. 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.</p>  
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<p>The T4 Bacteriophage uses a holin-endolysin system for host lysis. Endolysin, which has no secretory signal sequence, accumulates in a fully folded and active state in the cytosol. Holin, a late stage phage protein, triggers the degradation of the cell membrane allowing Endolysin to reach its target, the cell wall. In the phage genome, Endolysin is encoded by the e-gene and e<sup>-</sup> mutants have been shown to lack any noticeable lytic activity. Expression of holin without a corresponding endolysin results in complete and irreversible cessation of host metabolism without any structural damage to the outer membrane.<sup>[1]</sup></p></p>  
  
 
<h2>Usage</h2>
 
<h2>Usage</h2>
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<p>We used this part for screening of the e-gene lacking recombined phages after Lambda Red recombination. The T4 phage strains without the e-gene that codes for Endolysin were modified using homologous recombination to add mcp-1 attached to signal peptide under the t gene(codes for holin).The recomninant T4 phages give very small or no plaques on performing a plaque assay on the wild strains of <I>E. coli</I> as opposed to the wild type T4 phages. However on replica plating on the <I>E.coli</I> cells having the part transformed into them will give plaques with Wild type as well as the recombinant T4 phages. Thus we were able to select for the recombined phages.<sup>[1][2]</sup></p>
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<p>The 2018 IISc-Bangalore iGEM team used this part for screening of recombined phages lacking the e (endolysin) gene after Lambda Red recombination. The e-gene in the phage genome was replaced with a non-cognate protein and this part was used to screen for recombinants .The recombinant T4 phages give very small or no plaques on performing a plaque assay on the wild strains of <i>E. coli</i> (because of improper lysis) as opposed to the wild type T4 phages. However, replica plating of the plaques on <I>E.coli</I> cells having this part transformed into them allows easy identification by checking the plaques that have increased in size from one plate to another. The presence of recombinants can then be verified by a plaque PCR with primers for the non-cognate sequence.
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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>Expression and characterization</h3>
 
<h3>Expression and characterization</h3>
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<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>
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<p>The part was transformed into <I>E.coli</I> BL21(DE3) and an SDS-PAGE of the cell lysate was performed.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>[2]</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>
  
 
<h3>Characterisation of lystic activity of Endolysin </h3>
 
<h3>Characterisation of lystic activity of Endolysin </h3>
 
<img src="https://static.igem.org/mediawiki/parts/a/a2/Lytic_Activity_Characterisation_of_Endolysin_iGEM18_IISc_Bangalore.jpg" width=50% style="border: 1px solid black;" align="right">
 
<img src="https://static.igem.org/mediawiki/parts/a/a2/Lytic_Activity_Characterisation_of_Endolysin_iGEM18_IISc_Bangalore.jpg" width=50% style="border: 1px solid black;" align="right">
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<p>The lytic activity of endolysin was characterised by measuring the decrease in the optical density of B.cereus cell suspension after addition of endolysin<sup>[4][5]</sup>. Exponentially growing Bacillus cereus cells were washed twice and resuspended in 0.2mM Tris-HCl(pH-8) to adjust to OD<sub>600nm</sub>= 0.6-0.8. Then Endolysin protein(100 µL) was added to 900µL of cell suspension. The following were used as controls:<br>
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<p>The lytic activity of endolysin was characterised by measuring the decrease in the optical density of B.cereus cell suspension after addition of endolysin<sup>[3][4]</sup>. Exponentially growing Bacillus cereus cells were washed twice and resuspended in 0.2mM Tris-HCl(pH-8) to adjust the OD<sub>600nm</sub>= 0.6-0.8. Then Endolysin protein(100 µL) was added to 900µL of cell suspension. The following were used as controls:<br>
 
1. 900µL cell suspension with 100µL of resuspension buffer.<br>
 
1. 900µL cell suspension with 100µL of resuspension buffer.<br>
 
2. 900µL cell suspension with 100µL of Lysis Buffer used in protein extraction.<br>
 
2. 900µL cell suspension with 100µL of Lysis Buffer used in protein extraction.<br>
 
3. 900µL cell suspension wiht 100µL of Proteins from Wild Type E.coli(BL21(DE3))<br>
 
3. 900µL cell suspension wiht 100µL of Proteins from Wild Type E.coli(BL21(DE3))<br>
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The OD<sub>600nm</sub> at 37<sup>o</sup> 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.
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The Abs<sub>600nm</sub> at 37<sup>o</sup>C was measured using a plate reader at regular intervals of time and the lytic activity was quantified from the Change in %Abs v/s time curve as in Figure 2.
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<p>Addition of Endolysin has a much stronger lytic activity compared to the buffer alone. The higher activity of endolysin than the proteins extracted from the wild type led us to the conclusion that the lytic activity is conferred due to the Endolysin plasmid inserted.</p>
 
 
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K2609017 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_K2609002 parameters</partinfo>
 
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<!-- -->
 
  
 
===References===
 
===References===
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[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<br
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[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<br>
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[2]Wang, I N et al. “Holins: the protein clocks of bacteriophage infections.” Annual review of microbiology 54 (2000): 799-825.
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[2]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.<br>
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[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.<br>
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[3]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<br>
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[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<br>
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[4]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
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[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
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Latest revision as of 13:52, 11 October 2018

T4 endolysin under a constitutive promoter

This generator expresses the T4 endolysin protein (a phage lysozyme) under BBa_J23106, a medium strength constitutive promoter. Endolysin acts against the peptidoglycan layer and is minimally toxic to bacterial cells when produced internally because of the presence of an internal lipid membrane. An exposure of the peptidoglycan layer to endolysin however results in its disruption. Gram negative bacteria are resistant to lysis induced by external endolysin because of the presence of an outer membrane.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 311
    Illegal AgeI site found at 381
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Biology

The T4 Bacteriophage uses a holin-endolysin system for host lysis. Endolysin, which has no secretory signal sequence, accumulates in a fully folded and active state in the cytosol. Holin, a late stage phage protein, triggers the degradation of the cell membrane allowing Endolysin to reach its target, the cell wall. In the phage genome, Endolysin is encoded by the e-gene and e- mutants have been shown to lack any noticeable lytic activity. Expression of holin without a corresponding endolysin results in complete and irreversible cessation of host metabolism without any structural damage to the outer membrane.[1]

Usage

The 2018 IISc-Bangalore iGEM team used this part for screening of recombined phages lacking the e (endolysin) gene after Lambda Red recombination. The e-gene in the phage genome was replaced with a non-cognate protein and this part was used to screen for recombinants .The recombinant T4 phages give very small or no plaques on performing a plaque assay on the wild strains of E. coli (because of improper lysis) as opposed to the wild type T4 phages. However, replica plating of the plaques on E.coli cells having this part transformed into them allows easy identification by checking the plaques that have increased in size from one plate to another. The presence of recombinants can then be verified by a plaque PCR with primers for the non-cognate sequence.

Characterization

Expression and characterization

The part was transformed into E.coli BL21(DE3) and an SDS-PAGE of the cell lysate was performed.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[2]. We characterized the protein by checking for its lytic activity as mentioned below.

Characterisation of lystic activity of Endolysin

The lytic activity of endolysin was characterised by measuring the decrease in the optical density of B.cereus cell suspension after addition of endolysin[3][4]. Exponentially growing Bacillus cereus cells were washed twice and resuspended in 0.2mM Tris-HCl(pH-8) to adjust the 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 wiht 100µL of Proteins from Wild Type E.coli(BL21(DE3))
The Abs600nm at 37oC was measured using a plate reader at regular intervals of time and the lytic activity was quantified from the Change in %Abs v/s time curve as in Figure 2.


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]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.
[3]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
[4]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