Difference between revisions of "Part:BBa K3416000"
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=Introduction= | =Introduction= | ||
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| + | [[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. | ||
This part was used for the second goal- treatment - of the project FlavoFlow. | This part was used for the second goal- treatment - of the project FlavoFlow. | ||
| − | + | __TOC__ | |
| − | + | =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 < | + | 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. ''Trends in Microbiology'' '''28''', 633–643 (2020).</ref>. |
| − | AI-2 - interconverting molecules, which are derived from the same precursor and called the „universal“ bacterial signal < | + | AI-2 - interconverting molecules, which are derived from the same precursor and called the „universal“ bacterial signal <ref name="First" />, <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. ''BMC Evol Biol 4'', '''36''' (2004).</ref>, <ref>Xavier, K. B. & Bassler, B. L. Interference with AI-2-mediated bacterial cell-cell communication. ''Nature'' '''437''', 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>Xavier, K. B. et al. Phosphorylation and Processing of the Quorum-Sensing Molecule Autoinducer-2 in Enteric Bacteria. ''ACS Chem. Biol.'' '''2''', 128–136 (2007).</ref>. |
===Description of ''lsrACDBFG''=== | ===Description of ''lsrACDBFG''=== | ||
| − | ''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>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 E. coli. ''Metabolic Engineering'' '''12''', 291–297 (2010).</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>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'' gkw981 (2016) doi:10.1093/nar/gkw981.</ref>. |
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| + | ==Results== | ||
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| + | The results did not reiterate as expected. Only AI-2 inducible promoter <i>lsrACDBFG-sfGFP</i> signal showed a correlation with increasing AI-2 concentration(Fig.1 and Fig. 2). The EP01r is expected to be a stronger promoter than <i>lsrACDBFG</i>. To add more, all three AI-2 inducible promoters are leaky: when there is no AI-2 added there can be seen an emission of enhanced sfGFP. There is little known about these promoters. Thus, the conclusions are not valid enough. This might be affected by changing the plate reader devices, non-optimized sequences, or unprocessed experiment conditions. | ||
| + | However, after the last experiments construct <i>lsrACDBFG-mazF-J23117-sfGFP</i> showed positive results: there can be seen a positive toxin mazF activity. The increase of AI-2 concentration leads to cell-lysis of <i>E. coli</i>. | ||
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| + | <div><ul> | ||
| + | <li style="display: inline-block;"> [[File:T--Vilnius-Lithuania--ep01 sfGFP.png|thumb|none|300px|<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. ]] </li> | ||
| + | <li style="display: inline-block;"> [[File:T--Vilnius-Lithuania--LsrACDBFG sfGFP.png|thumb|none|300px|<b>Figure 2. </b> lsrACDBFG promoter strength measurement inducing with AI-2 after 6h of bacteria growth. From left to right: ‘+’- a positive control (J23117-sfGFP), without AI-2, 12μM, 18μM, 24μM of AI-2.]] </li> | ||
| + | </ul></div> | ||
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| + | <span class='h3bb'>Sequence and Features</span> | ||
| + | <partinfo>BBa_K3416000 SequenceAndFeatures</partinfo> | ||
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| + | <!-- Uncomment this to enable Functional Parameter display | ||
| + | ===Functional Parameters=== | ||
| + | <partinfo>BBa_K3416000 parameters</partinfo> | ||
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| + | =References= | ||
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Latest revision as of 18:01, 17 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 lsrACDBFG
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 results did not reiterate as expected. Only AI-2 inducible promoter lsrACDBFG-sfGFP signal showed a correlation with increasing AI-2 concentration(Fig.1 and Fig. 2). The EP01r is expected to be a stronger promoter than lsrACDBFG. To add more, all three AI-2 inducible promoters are leaky: when there is no AI-2 added there can be seen an emission of enhanced sfGFP. There is little known about these promoters. Thus, the conclusions are not valid enough. This might be affected by changing the plate reader devices, non-optimized sequences, or unprocessed experiment conditions. However, after the last experiments construct lsrACDBFG-mazF-J23117-sfGFP showed positive results: there can be seen a positive toxin mazF activity. The increase of AI-2 concentration leads to cell-lysis of E. coli.
Sequence and Features
- 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).
- ↑ Xavier, K. B. et al. Phosphorylation and Processing of the Quorum-Sensing Molecule Autoinducer-2 in Enteric Bacteria. ACS Chem. Biol. 2, 128–136 (2007).
- ↑ 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 E. coli. Metabolic Engineering 12, 291–297 (2010).
- ↑ 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 gkw981 (2016) doi:10.1093/nar/gkw981.