Difference between revisions of "Part:BBa K2136002"

Revision as of 21:13, 18 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).

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.
<img src=""

Figure 1. PCR from pSB1C3 + Lysostaphin

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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.
-Codon optimization for Chlamydomonas reinhardtii
-<html>
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-</head>
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-<table>
-  <tr>
-    <th><i>Staphylococcus simulans<i/></th>
-    <th><i>Chlamydomonas reinhardtii<i/></th>
-  </tr>
-  <tr>
-<td>GCTGCAACACATGAACATTCAGCACAATG
-GTTGAATAATTACAAAAAAGGATATGGTTACGG
-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
-GCTCAACAACTACAAGAAGGGCTACGGCTACGG
-TCCCTACCCGCTGGGGATTAACGGTGGCATGCA
-CTACGGCGTCGATTTCTTCATGAACATTGGCAC
-GCCCGTGAAGGCGATTAGCTCGGGCAAGATCGT
-CGAGGCTGGGTGGAGCAACTACGGTGGTGGGAA
-CCAGATCGGGCTGATCGAGAACGACGGGGTCCA
-CCGCCAGTGGTACATGCATCTCTCCAAGTACAA
-CGTCAAGGTGGGGGACTACGTCAAGGCGGGGCA
-GATTATTGGCTGGTCGGGCTCCACGGGGTACTC
-GACGGCCCCGCATCTCCATTTCCAGCGGATGGT
-CAACTCGTTCTCGAACTCGACCGCTCAGGACCC
-GATGCCGTTTCTGAAGTCGGCTGGGTACGGCAA
-GGCTGGGGGGACCGTCACCCCTACGCCTAACAC
-CGGGTGGAAGACCAACAAGTACGGGACGCTGTA
-CAAGTCCGAGTCGGCTAGCTTTACGCCTAACAC
-CGACATCATTACGCGGACCACGGGGCCGTTTCG
-CTCCATGCCGCAGAGCGGGGTCCTGAAGGCGGG
-CCAGACGATCCATTACGACGAGGTGATGAAGCA
-GGATGGTCATGTCTGGGTGGGTTACACCGGGAA
-CTCCGGCCAGCGCATCTACCTCCCCGTCCGGAC
-GTGGAACAAGTCCACCAACACGCTGGGGGTCCT
-GTGGGGGACGATCAAG</td>
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 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.
+<br>
 <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>
+</br>