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Team:UI-Indonesia/Project/Biofilm Degrading
From 2014.igem.org
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<h3>Biofilm Development in <i>V. cholerae </i></h3> | <h3>Biofilm Development in <i>V. cholerae </i></h3> | ||
<img width="550px" height="auto" align="right" style="margin-left:20px;margin-bottom:5px;clear:both;" src="https://static.igem.org/mediawiki/2014/f/f8/UI-Indonesia_Image_Header_BiofilmDevelopmentVCholerae.jpg" alt=""></img> | <img width="550px" height="auto" align="right" style="margin-left:20px;margin-bottom:5px;clear:both;" src="https://static.igem.org/mediawiki/2014/f/f8/UI-Indonesia_Image_Header_BiofilmDevelopmentVCholerae.jpg" alt=""></img> | ||
| - | <p><i>Vibrio cholerae</i> biofilm formation is performed to pass through stomach acid to colonize small and large intestine leading to hyperinfectious phenotype of <i>Vibrio cholerae</i><sup>[4]</sup></a>. The bacteria that become part of biofilm is united by quorum sensing signal. <i>Vibrio cholerae</i> possesses two pararel quorum signal molecule such as: <i><b>autoinducer 2 (Al-2)</b></i> (S-THMF-borate) is synthesized by LuxS and detected by LuxPQ, and <i><b>cholerae autoinducer 1 (CAI-1)</b></i> ((S)-3-hydroxytridecan-4-one) is made by CqsA and detected by <b>CqsS</b>. CAI-1 pathway has stronger influence on quorum-sensing regulated gene expression than Al-2 pathway. There are four Qrr sRNAs which activate regulator AphA in low cell density and repress regulator HapR leading to individual cells vice versa. In high cell density regulator HapR will be activated and cells begin grouping to form biofilm. During early stage of planktonic phase V. cholera will produce exopolysaccaharides and toxin co-ragulated pili enabling cells to attach and colonize the host and in the late phase of biofilm formation the cell will produce protease enabling cells exit and infect another site.<a href="">.</p> | + | <p><i>Vibrio cholerae</i> biofilm formation is performed to pass through stomach acid to colonize small and large intestine leading to hyperinfectious phenotype of <i>Vibrio cholerae</i><a href="https://2014.igem.org/Team:UI-Indonesia/Log_Book/References"><sup>[4]</sup></a>. The bacteria that become part of biofilm is united by quorum sensing signal. <i>Vibrio cholerae</i> possesses two pararel quorum signal molecule such as: <i><b>autoinducer 2 (Al-2)</b></i> (S-THMF-borate) is synthesized by LuxS and detected by LuxPQ, and <i><b>cholerae autoinducer 1 (CAI-1)</b></i> ((S)-3-hydroxytridecan-4-one) is made by CqsA and detected by <b>CqsS</b>. CAI-1 pathway has stronger influence on quorum-sensing regulated gene expression than Al-2 pathway. There are four Qrr sRNAs which activate regulator AphA in low cell density and repress regulator HapR leading to individual cells vice versa. In high cell density regulator HapR will be activated and cells begin grouping to form biofilm. During early stage of planktonic phase V. cholera will produce exopolysaccaharides and toxin co-ragulated pili enabling cells to attach and colonize the host and in the late phase of biofilm formation the cell will produce protease enabling cells exit and infect another site.<a href="https://2014.igem.org/Team:UI-Indonesia/Log_Book/References"><sup>[14]</sup></a>.</p> |
| - | <img width=" | + | <img width="800px" height="auto" align="center" style="margin:0px auto;margin-bottom:5px;clear:both;" src="https://static.igem.org/mediawiki/2014/4/4a/UI-Indonesia_Image_QuorumSensingVCho.jpg" alt=""></img> |
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<h2>Degrading Biofilm</h2> | <h2>Degrading Biofilm</h2> | ||
<h3>a) Alpha-amylase (α-amylase)</h3> | <h3>a) Alpha-amylase (α-amylase)</h3> | ||
| - | <p>Alpha amylase is an esential enzyme for α-glucan metabolism, functioned to catalyze hydrolysis α-1,4-glicosidic bond in glycogen, starch, and some others related polysaccharides | + | <p>Alpha amylase is an esential enzyme for α-glucan metabolism, functioned to catalyze hydrolysis α-1,4-glicosidic bond in glycogen, starch, and some others related polysaccharides<a href="https://2014.igem.org/Team:UI-Indonesia/Log_Book/References"><sup>[8]</sup></a>. MalS is gene responsible for <i>E. coli</i> periplasmic α-amylase strain K12, the length is 1967 bp<a href="https://2014.igem.org/Team:UI-Indonesia/Log_Book/References"><sup>[2]</sup></a>. When the quorum sensing signal is accepted by the CqsS receptor, it will activate transcription of MalS gene, and produce enzyme to degrade component of polysaccharides in biofilm of pathogenic bacteria.</p> |
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| + | <img width="800px" height="auto" align="center" style="margin:0px auto;margin-bottom:5px;clear:both;" src="https://static.igem.org/mediawiki/2014/7/7f/UI-Indonesia_Image_CircuitS100MalSHlyA.PNG" alt=""></img> | ||
| + | <br> | ||
MalS from previous part, <a href="http://parts.igem.org/Part:BBa_K523001">BBa_K523001</a> already have ribosome binding site in front of the gene so we do not put RBS again. Finally we construct a device consists of S100 promoter-RBS-MalS-HlyA (<a href="http://parts.igem.org/Part:BBa_K1344005">BBa_K1344005</a>) | MalS from previous part, <a href="http://parts.igem.org/Part:BBa_K523001">BBa_K523001</a> already have ribosome binding site in front of the gene so we do not put RBS again. Finally we construct a device consists of S100 promoter-RBS-MalS-HlyA (<a href="http://parts.igem.org/Part:BBa_K1344005">BBa_K1344005</a>) | ||
The HlyA is a signal peptide found in the C-terminal signal sequence of alpha-hemolysin (HlyA). Gram negative bacteria use this protein tag via the Type I secretion pathway. Fusion of the HlyA signal peptide to the target protein cause the excretion of protein to extracellular medium in a single step.</p> | The HlyA is a signal peptide found in the C-terminal signal sequence of alpha-hemolysin (HlyA). Gram negative bacteria use this protein tag via the Type I secretion pathway. Fusion of the HlyA signal peptide to the target protein cause the excretion of protein to extracellular medium in a single step.</p> | ||
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<br> | <br> | ||
<h3>b) Nuclease</h3> | <h3>b) Nuclease</h3> | ||
| - | <p>Nuclease is an extracellular enzyme which is able to cut phosphodiester bond between two unit nucleotides in nucleic acid, the length is 561 bp and Nuc is gene for nuclease synthesis. DNase is family of nuclease which is achieved from Staphylococcus aureus | + | <p>Nuclease is an extracellular enzyme which is able to cut phosphodiester bond between two unit nucleotides in nucleic acid, the length is 561 bp and Nuc is gene for nuclease synthesis. DNase is family of nuclease which is achieved from Staphylococcus aureus <a href="https://2014.igem.org/Team:UI-Indonesia/Log_Book/References"><sup>[15]</sup></a>. Here we make device consist of strong constitutive S100 promoter-RBS-Nuclease-HlyA (BBa_K1344003). Our device will be able to degrade extracellular DNA content of biofilm.</p> |
| - | <img width=" | + | <img width="800px" height="auto" align="center" style="margin:0px auto;margin-bottom:5px;clear:both;" src="https://static.igem.org/mediawiki/2014/7/76/UI-Indonesia_Image_CircuitS100RBSNucHlyA.PNG" alt=""></img> |
</div> | </div> | ||
Latest revision as of 03:27, 18 October 2014
Biofilm
Biofilm Composition
Biofilm Development in V. cholerae
Vibrio cholerae biofilm formation is performed to pass through stomach acid to colonize small and large intestine leading to hyperinfectious phenotype of Vibrio cholerae[4]. The bacteria that become part of biofilm is united by quorum sensing signal. Vibrio cholerae possesses two pararel quorum signal molecule such as: autoinducer 2 (Al-2) (S-THMF-borate) is synthesized by LuxS and detected by LuxPQ, and cholerae autoinducer 1 (CAI-1) ((S)-3-hydroxytridecan-4-one) is made by CqsA and detected by CqsS. CAI-1 pathway has stronger influence on quorum-sensing regulated gene expression than Al-2 pathway. There are four Qrr sRNAs which activate regulator AphA in low cell density and repress regulator HapR leading to individual cells vice versa. In high cell density regulator HapR will be activated and cells begin grouping to form biofilm. During early stage of planktonic phase V. cholera will produce exopolysaccaharides and toxin co-ragulated pili enabling cells to attach and colonize the host and in the late phase of biofilm formation the cell will produce protease enabling cells exit and infect another site.[14].
Degrading Biofilm
a) Alpha-amylase (α-amylase)
Alpha amylase is an esential enzyme for α-glucan metabolism, functioned to catalyze hydrolysis α-1,4-glicosidic bond in glycogen, starch, and some others related polysaccharides[8]. MalS is gene responsible for E. coli periplasmic α-amylase strain K12, the length is 1967 bp[2]. When the quorum sensing signal is accepted by the CqsS receptor, it will activate transcription of MalS gene, and produce enzyme to degrade component of polysaccharides in biofilm of pathogenic bacteria.
MalS from previous part, BBa_K523001 already have ribosome binding site in front of the gene so we do not put RBS again. Finally we construct a device consists of S100 promoter-RBS-MalS-HlyA (BBa_K1344005) The HlyA is a signal peptide found in the C-terminal signal sequence of alpha-hemolysin (HlyA). Gram negative bacteria use this protein tag via the Type I secretion pathway. Fusion of the HlyA signal peptide to the target protein cause the excretion of protein to extracellular medium in a single step.
b) Nuclease
Nuclease is an extracellular enzyme which is able to cut phosphodiester bond between two unit nucleotides in nucleic acid, the length is 561 bp and Nuc is gene for nuclease synthesis. DNase is family of nuclease which is achieved from Staphylococcus aureus [15]. Here we make device consist of strong constitutive S100 promoter-RBS-Nuclease-HlyA (BBa_K1344003). Our device will be able to degrade extracellular DNA content of biofilm.
