Difference between revisions of "Part:BBa K2136002"
 
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Lysostaphin is a 27 kDa-zinc metalloproteinase (EC 3.4.24.75) produced by Staphylococcus simulans. This enzyme hydrolyses glycine/glycine bonds in the pentaglycine interpeptide that maintain the stability of peptidoglycans in staphylococcal cell wall (Surovtsev 1).
 
Lysostaphin is a 27 kDa-zinc metalloproteinase (EC 3.4.24.75) produced by Staphylococcus simulans. This enzyme hydrolyses glycine/glycine bonds in the pentaglycine interpeptide that maintain the stability of peptidoglycans in staphylococcal cell wall (Surovtsev 1).
 +
 +
===Useful IDs ===
 +
*UniProtKB - P10547 (LSTP_STASI)
 +
*EC: 3.4.24.75
 +
*Corresponding Pfam domain: Peptidase_M23
 +
*Evidence: experimental (PubMed: 14317407)
 +
*PRIDE database: P10547.
  
 
===Usage===
 
===Usage===
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As our project relies on the advantage of Chlamydomonas reinhardtii as a proper chassis for high GC-content genes, our team adapted mature coding sequence of lysostaphin and codon optimized for microalgae. Notice that stop codon was removed from original sequence.
 
As our project relies on the advantage of Chlamydomonas reinhardtii as a proper chassis for high GC-content genes, our team adapted mature coding sequence of lysostaphin and codon optimized for microalgae. Notice that stop codon was removed from original sequence.
  
−
Codon optimization for Chlamydomonas reinhardtii
+
<p><i>Cloning into pSB1C3 vector</i></p>
 +
For further details, explore our complete experimental part!
 +
<br>Analytical digestion<br/>
 +
After several trials of transformation in DH-5α cells, plasmid bearing lysostaphin sequence was obtained. Analytical digestion was normally performed with about 500ng of plasmid.
 +
 
<html>
 
<html>
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<style>
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<img src="https://static.igem.org/mediawiki/parts/0/03/T--USP_UNIFESP-Brazil--Lysosdigestion.png" width="300px" style="margin-bottom:20px; margin-top:0px;" />
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table {
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<p class="fig-label"><b>Figure 1.</b> Analytical digestion pSB1C3 + Lysostaphin</p>
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    font-family: arial, sans-serif;
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</html>
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    border-collapse: collapse;
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    width: 50%;
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−
}
+
  
−
td, th {
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<br>PCR confirmation<br/>
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    border: 1px solid #dddddd;
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From plasmid backbone, PCR reaction was performed in order to identify the insert. Either our home-made X7 and Q5 polymerase worked perfectly.
−
    text-align: left;
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    padding: 12px;
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<html>
−
}
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<img src="https://static.igem.org/mediawiki/parts/9/93/T--USP_UNIFESP-Brazil--Lysosfromplasmid.png" width="300px" style="margin-bottom:20px; margin-top:0px;" />
 +
<p class="fig-label"><b>Figure 2.</b> PCR from pSB1C3 backbone </p>
 +
</html>
  
−
tr:nth-child(even) {
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<br>Sequencing<br/>
−
    background-color:;
+
Through the last months, lysostaphin feed our hopes and became one of our favorite parts. In order to confirm that BBa_K2136002 was cloned properly, sequencing was performed under standard protocol provided by local service.
−
}
+
−
</style>
+
−
</head>
+
−
<body>
+
  
−
<table>
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Sequencing with forward primer
−
  <tr>
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−
    <th><i>Staphylococcus simulans<i/></th>
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−
    <th><i>Chlamydomonas reinhardtii<i/></th>
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−
   
+
−
  </tr>
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−
  <tr>
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−
<td>GCTGCAACACATGAACATTCAGCACAATG
+
−
GTTGAATAATTACAAAAAAGGATATGGTTACGG
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−
TCCTTATCCATTAGGTATAAATGGCGGTATGCA
+
−
CTACGGAGTTGATTTTTTTATGAATATTGGAAC
+
−
ACCAGTAAAAGCTATTTCAAGCGGAAAAATAGT
+
−
TGAAGCTGGTTGGAGTAATTACGGAGGAGGTAA
+
−
TCAAATAGGTCTTATTGAAAATGATGGAGTGCA
+
−
TAGACAATGGTATATGCATCTAAGTAAATATAA
+
−
TGTTAAAGTAGGAGATTATGTCAAAGCTGGTCA
+
−
AATAATCGGTTGGTCTGGAAGCACTGGTTATTC
+
−
TACAGCACCACATTTACACTTCCAAAGAATGGT
+
−
TAATTCATTTTCAAATTCAACTGCCCAAGATCC
+
−
AATGCCTTTCTTAAAGAGCGCAGGATATGGAAA
+
−
AGCAGGTGGTACAGTAACTCCAACGCCGAATAC
+
−
AGGTTGGAAAACAAACAAATATGGCACACTATA
+
−
TAAATCAGAGTCAGCTAGCTTCACACCTAATAC
+
−
AGATATAATAACAAGAACGACTGGTCCATTTAG
+
−
AAGCATGCCGCAGTCAGGAGTCTTAAAAGCAGG
+
−
TCAAACAATTCATTATGATGAAGTGATGAAACA
+
−
AGACGGTCATGTTTGGGTAGGTTATACAGGTAA
+
−
CAGTGGCCAACGTATTTACTTGCCTGTAAGAAC
+
−
ATGGAATAAATCTACTAATACTTTAGGTGTTCT
+
−
TTGGGGAACTATAAAG<b>TGA (STOP CODON) </b></td>
+
  
−
<td>GCTGCTACCCACGAGCATTCCGCCCAGTG
+
<html>
−
GCTCAACAACTACAAGAAGGGCTACGGCTACGG
+
<center><img src="https://static.igem.org/mediawiki/parts/4/4d/T--USP_UNIFESP-Brazil--Sequencingforward.png" width="800px" style="margin-bottom:20px; margin-top:0px;" /></center>
−
TCCCTACCCGCTGGGGATTAACGGTGGCATGCA
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<center><p class="fig-label"><b>Figure 3.</b> Sequencing reaction for forward primer</p></center>
−
CTACGGCGTCGATTTCTTCATGAACATTGGCAC
+
</html>
−
GCCCGTGAAGGCGATTAGCTCGGGCAAGATCGT
+
−
CGAGGCTGGGTGGAGCAACTACGGTGGTGGGAA
+
−
CCAGATCGGGCTGATCGAGAACGACGGGGTCCA
+
−
CCGCCAGTGGTACATGCATCTCTCCAAGTACAA
+
−
CGTCAAGGTGGGGGACTACGTCAAGGCGGGGCA
+
−
GATTATTGGCTGGTCGGGCTCCACGGGGTACTC
+
−
GACGGCCCCGCATCTCCATTTCCAGCGGATGGT
+
−
CAACTCGTTCTCGAACTCGACCGCTCAGGACCC
+
−
GATGCCGTTTCTGAAGTCGGCTGGGTACGGCAA
+
−
GGCTGGGGGGACCGTCACCCCTACGCCTAACAC
+
−
CGGGTGGAAGACCAACAAGTACGGGACGCTGTA
+
−
CAAGTCCGAGTCGGCTAGCTTTACGCCTAACAC
+
−
CGACATCATTACGCGGACCACGGGGCCGTTTCG
+
−
CTCCATGCCGCAGAGCGGGGTCCTGAAGGCGGG
+
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CCAGACGATCCATTACGACGAGGTGATGAAGCA
+
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GGATGGTCATGTCTGGGTGGGTTACACCGGGAA
+
−
CTCCGGCCAGCGCATCTACCTCCCCGTCCGGAC
+
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GTGGAACAAGTCCACCAACACGCTGGGGGTCCT
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GTGGGGGACGATCAAG</td>  
+
  
−
<p><i>Cloning into pSB1C3 vector</i></p>
+
Sequencing with reverse primer
−
For further details, explore our complete experimental part!
+
  
−
Analytical digestion
+
<html>
−
After several trials of transformation in DH-5α cells, plasmid bearing lysostaphin sequence was obtained. Analytical digestion was normally performed with about 500ng of plasmid.
+
<center><img src="https://static.igem.org/mediawiki/parts/7/70/T--USP_UNIFESP-Brazil--Sequencingreverse.png" width="800px" style="margin-bottom:20px; margin-top:0px;" /></center>
 +
<center><p class="fig-label"><b>Figure 4.</b> Sequencing reaction for reverse primer</p></center>
  
−
<img src="https://static.igem.org/mediawiki/2016/9/93/T--USP_UNIFESP-Brazil--Lysosfromplasmid.png"
 
−
<p class="fig-label"> <b>Figure 1.</b> PCR from pSB1C3 + Lysostaphin </p>
 
  
 +
 +
<html>
 +
<div class="small-10 columns small-offset-2">
 +
<div class="small-10 small-offset-1 columns">
 +
<p class="black"> For further details of sequencing data, check <a href= https://static.igem.org/mediawiki/2016/d/df/T--USP_UNIFESP-Brazil--sequencingdata.zip> here!</a>
 +
                              </p>
 +
</html>
 +
 +
<p><b>References</b></p>
 +
<div class="small-10 columns small-offset-2">
 +
<div class="small-10 small-offset-1 columns">
 +
<p class="black">Baker, Christopher C., Carol L. Miller, and DONALD D. TRUNKEY. "Predicting fatal sepsis in burn patients." Journal of Trauma and Acute Care Surgery 19.9 (1979): 641-648.</p>
 +
<p class="black">Hojckova, Katarina, Matej Stano, and Lubos Klucar. "phiBIOTICS: catalogue of therapeutic enzybiotics, relevant research studies and practical applications." BMC microbiology 13.1 (2013): 1.</p>
 +
<p class="black">Leseva, M., et al. "Nosocomial infections in burn patients: etiology, antimicrobial resistance, means to control." Ann Burns Fire Disasters 26.1 (2013): 5-11.</p>
 +
<p class="black">Kumar, Jaspal K. "Lysostaphin: an antistaphylococcal agent." Applied microbiology and biotechnology 80.4 (2008): 555-561.</p>
 +
<p class="black">Hardy J, Römer L, Scheibel T (2008) Polymeric materials based on silk proteins. Polymer 49 (20): 4309-4327. DOI: 10.1016/j.polymer.2008.08.006</p>
 +
<p class="black">Kluge J, Rabotyagova O, Leisk G, Kaplan D (2008) Spider silks and their applications. Trends in Biotechnology 26 (5): 244-251. DOI: 10.1016/j.tibtech.2008.02.006</p>
 +
<p class="black">Lewis R (2006) Spider Silk: Ancient Ideas for New Biomaterials. Chemical Reviews 106 (9): 3762-3774. DOI: 10.1021/cr010194g</p>
 +
<p class="black">Pavoni, Vittorio, et al. "Outcome predictors and quality of life of severe burn patients admitted to intensive care unit." Scandinavian journal of trauma, resuscitation and emergency medicine 18.1 (2010): 1.</p>
 +
<p class="black">Surovtsev, V. I., et al. "Ionogenic groups in the active site of lysostaphin. Kinetic and thermodynamic data compared with X-ray crystallographic data."Biochemistry (Moscow) 72.9 (2007): 989-993</p>
 +
 +
</div>
 +
</div>
  
  

Latest revision as of 07:32, 19 October 2016


Lysostaphin

Lysostaphin is a 27 kDa-zinc metalloproteinase (EC 3.4.24.75) produced by Staphylococcus simulans. This enzyme hydrolyses glycine/glycine bonds in the pentaglycine interpeptide that maintain the stability of peptidoglycans in staphylococcal cell wall (Surovtsev 1).

Useful IDs

  • UniProtKB - P10547 (LSTP_STASI)
  • EC: 3.4.24.75
  • Corresponding Pfam domain: Peptidase_M23
  • Evidence: experimental (PubMed: 14317407)
  • PRIDE database: P10547.

Usage

In spite of recent advances in medical care, data reveals that mortality rate among burned patients remains higher depending on the extent of affected area (Pavoni 2). These death rates could also be driven by prolonged antibiotic courses, which related to immunocompetence decrease. Consequently, leading patient to a vulnerability state of the skin to microorganisms (Baker 3), besides the per se lost of the first line of antimicrobial defense. Given the fact that hospital acquired infections is a growing concern in public health alongside the development of resistant strains, burned individuals are at the risk top risk among patients (Leseva 4).

Biology

Lysostaphin is encoded by a three-modular gene that encompasses a self-cleavage peptide, a propetide and the self-coding sequence of lysostaphin. After protein processing, mature lysostaphin (246 aminoacids) is released and exhibits a triple-enzymatic activity: glycylglycine endopeptidase, endo-β-N-acetyl glucosamidase and N-acetyl-muramyl-L-alanine. Consequently, it rapidly lyses actively growing and non-dividing cells including staphylococci in biofilms and, due to its specificity, it could have high potential in the treatment of antibiotic-resistant staphylococcal infections (Kumar 5).

Perspectives

This protein with antibiotic properties seems as a promissory candidate for medical purposes that combined with novel chimeric spider silk as polymeric matrix could become an interesting approach to address nosocomial infections by resistant strains.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 505
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


It’s important to take into account that this sequence encompasses only mature lysostaphin.

As our project relies on the advantage of Chlamydomonas reinhardtii as a proper chassis for high GC-content genes, our team adapted mature coding sequence of lysostaphin and codon optimized for microalgae. Notice that stop codon was removed from original sequence.

Cloning into pSB1C3 vector

For further details, explore our complete experimental part!
Analytical digestion
After several trials of transformation in DH-5α cells, plasmid bearing lysostaphin sequence was obtained. Analytical digestion was normally performed with about 500ng of plasmid.

Figure 1. Analytical digestion pSB1C3 + Lysostaphin


PCR confirmation
From plasmid backbone, PCR reaction was performed in order to identify the insert. Either our home-made X7 and Q5 polymerase worked perfectly.

Figure 2. PCR from pSB1C3 backbone


Sequencing
Through the last months, lysostaphin feed our hopes and became one of our favorite parts. In order to confirm that BBa_K2136002 was cloned properly, sequencing was performed under standard protocol provided by local service.

Sequencing with forward primer

Figure 3. Sequencing reaction for forward primer

Sequencing with reverse primer

Figure 4. Sequencing reaction for reverse primer

For further details of sequencing data, check here!

References

Baker, Christopher C., Carol L. Miller, and DONALD D. TRUNKEY. "Predicting fatal sepsis in burn patients." Journal of Trauma and Acute Care Surgery 19.9 (1979): 641-648.

Hojckova, Katarina, Matej Stano, and Lubos Klucar. "phiBIOTICS: catalogue of therapeutic enzybiotics, relevant research studies and practical applications." BMC microbiology 13.1 (2013): 1.

Leseva, M., et al. "Nosocomial infections in burn patients: etiology, antimicrobial resistance, means to control." Ann Burns Fire Disasters 26.1 (2013): 5-11.

Kumar, Jaspal K. "Lysostaphin: an antistaphylococcal agent." Applied microbiology and biotechnology 80.4 (2008): 555-561.

Hardy J, Römer L, Scheibel T (2008) Polymeric materials based on silk proteins. Polymer 49 (20): 4309-4327. DOI: 10.1016/j.polymer.2008.08.006

Kluge J, Rabotyagova O, Leisk G, Kaplan D (2008) Spider silks and their applications. Trends in Biotechnology 26 (5): 244-251. DOI: 10.1016/j.tibtech.2008.02.006

Lewis R (2006) Spider Silk: Ancient Ideas for New Biomaterials. Chemical Reviews 106 (9): 3762-3774. DOI: 10.1021/cr010194g

Pavoni, Vittorio, et al. "Outcome predictors and quality of life of severe burn patients admitted to intensive care unit." Scandinavian journal of trauma, resuscitation and emergency medicine 18.1 (2010): 1.

Surovtsev, V. I., et al. "Ionogenic groups in the active site of lysostaphin. Kinetic and thermodynamic data compared with X-ray crystallographic data."Biochemistry (Moscow) 72.9 (2007): 989-993