Difference between revisions of "Part:BBa K3416001"
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| + | __NOTOC__ | ||
=Introduction= | =Introduction= | ||
| − | [[File:T--Vilnius-Lithuania--FFlogo.png| | + | [[File:T--Vilnius-Lithuania--FFlogo.png|100px|right|FlavoFlow]] |
Vilnius-Lithuania iGEM 2020 project [https://2020.igem.org/Team:Vilnius-Lithuania <b>FlavoFlow]</b>includes three goals towards looking for <i>Flavobacterium</i> disease-related problems’ solutions. The project includes creating a rapid detection kit, based on HDA and LFA, developing an implement for treating a disease, and introducing the foundation of edible vaccines. | Vilnius-Lithuania iGEM 2020 project [https://2020.igem.org/Team:Vilnius-Lithuania <b>FlavoFlow]</b>includes three goals towards looking for <i>Flavobacterium</i> disease-related problems’ solutions. The project includes creating a rapid detection kit, based on HDA and LFA, developing an implement for treating a disease, and introducing the foundation of edible vaccines. | ||
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[[File:T--Vilnius-Lithuania--flavoflowlogo.png|200px|right|FlavoFlow]] | [[File:T--Vilnius-Lithuania--flavoflowlogo.png|200px|right|FlavoFlow]] | ||
| − | + | __TOC__ | |
| − | + | =Biology= | |
| − | AI-2 - interconverting molecules, which are derived from the same precursor and called the „universal“ bacterial signal < | + | Quorum sensing (QS) is a communication system, which controls gene expression in response to population density, between bacteria. There are two QS systems: the first one, based on AI-1 or acyl-homoserine lactone (AHL), and the second – autoinducer 2 <ref name= "First">Stephens, K. & Bentley, W. E. Synthetic Biology for Manipulating Quorum Sensing in Microbial Consortia. <i>Trends in Microbiology</i> <b>28</b>, 633–643 (2020).</ref>. |
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| + | AI-2 - interconverting molecules, which are derived from the same precursor and called the „universal“ bacterial signal <ref name= "First"></ref>, <ref>Sun, J., Daniel, R., Wagner-Döbler, I. & Zeng, A.-P. Is autoinducer-2 a universal signal for interspecies communication: a comparative genomic and phylogenetic analysis of the synthesis and signal transduction pathways. <i>BMC Evol Biol</i> <b>4</b>, 36 (2004).</ref>, <ref>Xavier, K. B. & Bassler, B. L. Interference with AI-2-mediated bacterial cell-cell communication. <i>Nature</i> <b>437</b>, 750–753 (2005).</ref>. AI-2 controls the expression of LuxS regulated transporter, which is responsible for incorporation, phosphorylating, and processing of the AI-2 signal. The lsr transporter has genes, which expression is regulated by AI-2. LsrR is a repressor of the lsr operon. The AI-2, phosphorylated by lsrK, leads to derepression of lsr operon <ref>Pei, D. & Zhu, J. Mechanism of action of S-ribosylhomocysteinase (LuxS). <i>Current Opinion in Chemical Biology</i> <b>8</b>, 492–497 (2004).</ref>. | ||
===Description of EP01rec=== | ===Description of EP01rec=== | ||
| − | ''lsrACDBFGE'' is an operon that regulates the genes' expression involved in AI-2 uptake and degradation. Autoinducer 2 (AI-2) is phosphorylated by LsrK, kinase, to phospho-AI-2. The phospho-AI-2 de-represses the ''lsrACDBFGE'' operon‘s repressor LsrR resulting in the induction of the genes < | + | ''lsrACDBFGE'' is an operon that regulates the genes' expression involved in AI-2 uptake and degradation. Autoinducer 2 (AI-2) is phosphorylated by LsrK, kinase, to phospho-AI-2. The phospho-AI-2 de-represses the ''lsrACDBFGE'' operon‘s repressor LsrR resulting in the induction of the genes <ref>Hauk, P. <i>et al.</i> Insightful directed evolution of <i>Escherichia coli</i> quorum sensing promoter region of the lsrACDBFG operon: a tool for synthetic biology systems and protein expression. <i>Nucleic Acids Res<i> gkw981 (2016) doi:10.1093/nar/gkw981.</ref>. Hauk and her colleagues created promoters library from ''lsrACDBFG'' operon region. They have achieved two lsrACDBFG mutants, '''EP01rec''' and EP14rec, which has the same as WT function and successfully evolved <ref>Tsao, C.-Y., Hooshangi, S., Wu, H.-C., Valdes, J. J. & Bentley, W. E. Autonomous induction of recombinant proteins by minimally rewiring native quorum sensing regulon of <i>E. coli</i>. <i>Metabolic Engineering</i> <b>12</b>, 291–297 (2010).</ref>. |
| + | |||
| + | ==Results== | ||
| + | |||
| + | The EP01r is expected to be a stronger promoter than <i>lsrACDBFG</i>, however, after EP01 promoter strength measurements there was seen that EP01 promoter is weaker than <i>lsrACDBFG</i>. | ||
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| + | {| align=left | ||
| + | |- | ||
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| + | [[File:T--Vilnius-Lithuania--ep01 sfGFP.png|thumb|left|<b>Figure 1. </b> EP01r promoter strength measurement inducing with AI-2. From left to right: ‘+’- a positive control (J23117-sfGFP), without AI-2, 12μM, 18μM, 24μM of AI-2.]] | ||
| + | |- | ||
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| + | [[File:T--Vilnius-Lithuania--LsrACDBFG lineplot.png|thumb|left|<b>Figure 2. </b> lsrACDFBG promoter strength measurement inducing by AI-2 changes over time. The concentrations varied from 0μM to 24μM.]] | ||
| + | |} | ||
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| + | <!-- --> | ||
| + | <span class='h3bb'>Sequence and Features</span> | ||
| + | <partinfo>BBa_K3416001 SequenceAndFeatures</partinfo> | ||
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| + | <!-- Uncomment this to enable Functional Parameter display | ||
| + | ===Functional Parameters=== | ||
| + | <partinfo>BBa_K3416001 parameters</partinfo> | ||
| + | <!-- --> | ||
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| + | =References= | ||
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Revision as of 21:19, 16 December 2020
Introduction
Vilnius-Lithuania iGEM 2020 project FlavoFlowincludes three goals towards looking for Flavobacterium disease-related problems’ solutions. The project includes creating a rapid detection kit, based on HDA and LFA, developing an implement for treating a disease, and introducing the foundation of edible vaccines. This part was used for the second goal- treatment - of the project FlavoFlow.
Biology
Quorum sensing (QS) is a communication system, which controls gene expression in response to population density, between bacteria. There are two QS systems: the first one, based on AI-1 or acyl-homoserine lactone (AHL), and the second – autoinducer 2 [1].
AI-2 - interconverting molecules, which are derived from the same precursor and called the „universal“ bacterial signal [1], [2], [3]. AI-2 controls the expression of LuxS regulated transporter, which is responsible for incorporation, phosphorylating, and processing of the AI-2 signal. The lsr transporter has genes, which expression is regulated by AI-2. LsrR is a repressor of the lsr operon. The AI-2, phosphorylated by lsrK, leads to derepression of lsr operon [4].
Description of EP01rec
lsrACDBFGE is an operon that regulates the genes' expression involved in AI-2 uptake and degradation. Autoinducer 2 (AI-2) is phosphorylated by LsrK, kinase, to phospho-AI-2. The phospho-AI-2 de-represses the lsrACDBFGE operon‘s repressor LsrR resulting in the induction of the genes [5]. Hauk and her colleagues created promoters library from lsrACDBFG operon region. They have achieved two lsrACDBFG mutants, EP01rec and EP14rec, which has the same as WT function and successfully evolved [6].
Results
The EP01r is expected to be a stronger promoter than lsrACDBFG, however, after EP01 promoter strength measurements there was seen that EP01 promoter is weaker than lsrACDBFG.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
- ↑ 1.0 1.1 Stephens, K. & Bentley, W. E. Synthetic Biology for Manipulating Quorum Sensing in Microbial Consortia. Trends in Microbiology 28, 633–643 (2020).
- ↑ Sun, J., Daniel, R., Wagner-Döbler, I. & Zeng, A.-P. Is autoinducer-2 a universal signal for interspecies communication: a comparative genomic and phylogenetic analysis of the synthesis and signal transduction pathways. BMC Evol Biol 4, 36 (2004).
- ↑ Xavier, K. B. & Bassler, B. L. Interference with AI-2-mediated bacterial cell-cell communication. Nature 437, 750–753 (2005).
- ↑ Pei, D. & Zhu, J. Mechanism of action of S-ribosylhomocysteinase (LuxS). Current Opinion in Chemical Biology 8, 492–497 (2004).
- ↑ Hauk, P. et al. Insightful directed evolution of Escherichia coli quorum sensing promoter region of the lsrACDBFG operon: a tool for synthetic biology systems and protein expression. Nucleic Acids Res<i> gkw981 (2016) doi:10.1093/nar/gkw981.</span>
</li>
- ↑ Tsao, C.-Y., Hooshangi, S., Wu, H.-C., Valdes, J. J. & Bentley, W. E. Autonomous induction of recombinant proteins by minimally rewiring native quorum sensing regulon of <i>E. coli</i>. <i>Metabolic Engineering</i> 12, 291–297 (2010).
</ol>