Difference between revisions of "Part:BBa K3416201"

 
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<partinfo>BBa_K3416201 short</partinfo>
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=Introduction=
  
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qwwe
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[[File:T--Vilnius-Lithuania--FFlogo.png|100px|right|FlavoFlow]]
  
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<!-- Add more about the biology of this part here
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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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===Usage and Biology===
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This part was used for the second goal- treatment -  of the project FlavoFlow.
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[[File:T--Vilnius-Lithuania--flavoflowlogo.png|200px|right|FlavoFlow]]
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__TOC__
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=Biology=
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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>.
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===Description of EP01rec===
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''lsrACDBFGE'' is an operon that regulates the genes&apos; 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>.
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==Results==
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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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[[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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<partinfo>BBa_K3416201 parameters</partinfo>
 
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=References=
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Revision as of 17:31, 17 December 2020


Introduction

FlavoFlow

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.

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.

Figure 1. 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.
Figure 2. lsrACDFBG promoter strength measurement inducing by AI-2 changes over time. The concentrations varied from 0μM to 24μM.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 642
    Illegal NheI site found at 665
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 743
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

  1. ↑ 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).
  2. ↑ 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).
  3. ↑ Xavier, K. B. & Bassler, B. L. Interference with AI-2-mediated bacterial cell-cell communication. Nature 437, 750–753 (2005).
  4. ↑ Pei, D. & Zhu, J. Mechanism of action of S-ribosylhomocysteinase (LuxS). Current Opinion in Chemical Biology 8, 492–497 (2004).
  5. ↑ 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.
  6. ↑ 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).