Difference between revisions of "Part:BBa K4719009"
 
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<partinfo>BBa_K4719009 short</partinfo>
 
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===Introduction===
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<partinfo>BBa_K4719009 SequenceAndFeatures</partinfo>
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Vilnius Lithuania iGEM 2023 team's goal was to create a universal synthetic biology system in ''Komagataeibacter xylinus'' for ''in vivo'' bacterial cellulose polymer composition modification. As a second approach, we designed indigo-dyed cellulose that could be used as a green chemistry way to apply cellulose in the textile industry.
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Colorful cellulose was made by introducing styrene monooxygenase pKARA_RT3 [https://parts.igem.org/Part:BBa_K4719018 BBa_K4719018] to ''K. xylinus''. This enzyme can metabolize indole and its other derivatives into indigo dyes. Bacteria produce cellulose alongside pigments. Since they are not water soluble, the final product retains the color.
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===Usage and Biology===
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<body>
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<h2>Introduction</h2>
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<p>
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<b>Vilnius-Lithuania iGEM 2023</b> team's goal was to create <b> synthetic biology tools for <i>in vivo</i> alterations of <i>Komagataeibacter xylinus</i> bacterial cellulose polymer composition</b>. Firstly, we chose to produce a <b>cellulose-chitin copolymer</b> that would later be deacetylated, creating <b>bacterial cellulose-chitosan</b>. This polymer is an easily modifiable platform when compared to bacterial cellulose. The enhanced chemical reactivity of the bacterial cellulose-chitosan polymer allows for specific functionalizations in the biomedicine field, such as scaffold design. As a second approach, we designed <b>indigo-dyed cellulose</b> that could be used as a green chemistry way to apply cellulose in the textile industry. Lastly, we have achieved a of <b>bacterial cellulose and polyhydroxybutyrate (PHB) composite</b>, which is synthesized by <i>K. xylinus</i>.
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</p>
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<p>
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Colorful cellulose was made by introducing styrene monooxygenase pKARA_RT3 <a href="https://parts.igem.org/Part:BBa_K4719018"> BBa_K4719018</a> to <i>K. xylinus</i>. This enzyme can metabolize indole and its other derivatives into indigo dyes. Bacteria produce cellulose alongside pigments. Since they are not water soluble, the final product retains the color.
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</p>
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<h2>Usage and Biology</h2>
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<p>
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pKARA_RT3 is styrene monooxygenase. Styrene monooxygenases belong to oxidoreductases, specifically to an enzyme class 1.14.14-, which act on paired donors, with incorporation or reduction of molecular oxygen, which is donated by reduced flavin or flavoprotein. Styrene monooxygenases have been shown to produce indigo - a blue dye [1]. Cells containing this enzyme are able to act as biocatalysts and convert indole to indigo. pKARA_RT3 is a basic part in <a href="https://parts.igem.org/Part:BBa_K4719018"> BBa_K4719018</a>, this construct is used for the production of colorful cellulose.
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</p>
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<h2>Characterisation</h2>
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<p>
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The sequence for pKARA_RT3 has been found by metagenomic analysis of a soil sample. To better understand the origin of this protein, we performed NCBI nucleotide BLAST analysis and with 99.11% identity, it was identified to be of Arthrobacter sp. StoSoilB22 DNA (Sequence ID: AP024652.1).
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</p>
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<figure>
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<div class = "center">
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<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/bba-k4719009-pkara-rt3-nucleotide-blast-dot.png" style = "width:400px;"></center>
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</div>
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<figcaption><center>Figure 1: Dot plot of pKARA_RT3 nucleotide sequence against Arthrobacter sp. StoSoilB22 DNA </center></figcaption>
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</figure>
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<p>
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For a better understanding of the function pKARA_RT3, we analyzed the amino acid sequence with UniProt BLAST analysis. The results showed with 93.2% identity to be styrene monooxygenase StyA putative substrate binding domain-containing protein (primary accesion A0A4Y3NB75). This result was consistent with the experimental data of the activity of pKARA_RT3 to metabolize indigo to indole compounds.
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</p>
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<figure>
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<div class = "center">
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<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/pkara-rt3-protein-blast-dot-plot-geras.png" style = "width:400px;"></center>
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</div>
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<figcaption><center>Figure 2: Dot plot of nucleotide sequence of pKARA_RT3 against nucleotide sequence of Styrene monooxygenase.</center></figcaption>
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</figure>
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<p>
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For further verification of pKARA_RT3 structure, we employed NCBI Domain architecture search. The two structures this tool identified were NAD-binding domain and a Fe-S cluster, as well Styrene monooxygenase A putative substrate binding domain.
 +
The results obtained by three different analysis were consistent and we were able to confirm the origin and function of the pKARA_RT3 protein.
 +
</p>
  
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pKARA_RT3 is styrene monooxygenase. Styrene monooxygenases belong to oxidoreductases, specifically to an enzyme class 1.14.14-, which act on paired donors, with incorporation or reduction of molecular oxygen, which is donated by reduced flavin or flavoprotein. Styrene monooxygenases have been shown to produce indigo - a blue dye [https://parts.igem.org/Part:BBa_K4719009#references (1)]. Cells containing this enzyme are able to act as biocatalysts and convert indole to indigo. pKARA_RT3 is a basic part in [https://parts.igem.org/Part:BBa_K4719018 BBa_K4719018], this construct is used for the production of colorful cellulose.
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===Characterisation===
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The sequence for pKARA_RT3 has been found by metagenomic analysis of a soil sample. To better understand the origin of this protein, we performed NCBI nucleotide BLAST analysis and with 99.11% identity, it was identified to be of Arthrobacter sp. StoSoilB22 DNA (Sequence ID: AP024652.1).
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[[https://static.igem.wiki/teams/4719/wiki/partai/bba-k4719009-pkara-rt3-nucleotide-blast-dot.png]|500px|thumb|left|Figure1: Dot plot of nucleotide sequence of pKARA_RT3 and identified sequence from Arthrobacter sp. StoSoilB22 DNA]]
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For a better understanding of the function pKARA_RT3, we analyzed the amino acid sequence with UniProt BLAST analysis. The results showed with 93.2% identity to be Styrene monooxygenase StyA putative substrate binding domain-containing protein. This result was consistent with the experimental data of the activity of pKARA_RT3 to metabolize indigo to indole compounds.
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[[Image:pkara-rt3-protein-blast-dot-plot.png|500px|thumb|left|Figure1: Dot plot of nucleotide sequences of pKARA_RT3 and Styrene monooxygenase StyA putative substrate binding domain-containing protein]]
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For further verification of pKARA_RT3 structure, we employed NCBI Domain architecture search. The two structures this tool identified were NAD-binding domain and a Fe-S cluster, as well Styrene monooxygenase A putative substrate binding domain.
+
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The results obtained by three different analysis were consistent and we were able to confirm the origin and function of the pKARA_RT3 protein.
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===Experimental characterisation===
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===References===
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<h3>References</h3>
 
1. O’Connor, K.E., Dobson, A.D. and Hartmans, S. (1997) ‘Indigo formation by microorganisms expressing styrene monooxygenase activity’, Applied and Environmental Microbiology, 63(11), pp. 4287–4291. doi:10.1128/aem.63.11.4287-4291.1997.  
 
1. O’Connor, K.E., Dobson, A.D. and Hartmans, S. (1997) ‘Indigo formation by microorganisms expressing styrene monooxygenase activity’, Applied and Environmental Microbiology, 63(11), pp. 4287–4291. doi:10.1128/aem.63.11.4287-4291.1997.  
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4719009 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4719009 SequenceAndFeatures</partinfo>
  
  
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<!-- Uncomment this to enable Functional Parameter display
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<h3>Functional Parameters</h3>
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===Functional Parameters===
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<partinfo>BBa_K4719009 parameters</partinfo>  
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<partinfo>BBa_K4719009 parameters</partinfo>
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Latest revision as of 21:01, 21 July 2024

pKARA_RT3 styrene monooxigenase


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1171
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1171
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1171
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1171
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1171
    Illegal NgoMIV site found at 37
    Illegal NgoMIV site found at 79
    Illegal NgoMIV site found at 448
    Illegal AgeI site found at 634
  • 1000
    COMPATIBLE WITH RFC[1000]


Introduction

Vilnius-Lithuania iGEM 2023 team's goal was to create synthetic biology tools for in vivo alterations of Komagataeibacter xylinus bacterial cellulose polymer composition. Firstly, we chose to produce a cellulose-chitin copolymer that would later be deacetylated, creating bacterial cellulose-chitosan. This polymer is an easily modifiable platform when compared to bacterial cellulose. The enhanced chemical reactivity of the bacterial cellulose-chitosan polymer allows for specific functionalizations in the biomedicine field, such as scaffold design. As a second approach, we designed indigo-dyed cellulose that could be used as a green chemistry way to apply cellulose in the textile industry. Lastly, we have achieved a of bacterial cellulose and polyhydroxybutyrate (PHB) composite, which is synthesized by K. xylinus.

Colorful cellulose was made by introducing styrene monooxygenase pKARA_RT3 BBa_K4719018 to K. xylinus. This enzyme can metabolize indole and its other derivatives into indigo dyes. Bacteria produce cellulose alongside pigments. Since they are not water soluble, the final product retains the color.

Usage and Biology

pKARA_RT3 is styrene monooxygenase. Styrene monooxygenases belong to oxidoreductases, specifically to an enzyme class 1.14.14-, which act on paired donors, with incorporation or reduction of molecular oxygen, which is donated by reduced flavin or flavoprotein. Styrene monooxygenases have been shown to produce indigo - a blue dye [1]. Cells containing this enzyme are able to act as biocatalysts and convert indole to indigo. pKARA_RT3 is a basic part in BBa_K4719018, this construct is used for the production of colorful cellulose.

Characterisation

The sequence for pKARA_RT3 has been found by metagenomic analysis of a soil sample. To better understand the origin of this protein, we performed NCBI nucleotide BLAST analysis and with 99.11% identity, it was identified to be of Arthrobacter sp. StoSoilB22 DNA (Sequence ID: AP024652.1).

Figure 1: Dot plot of pKARA_RT3 nucleotide sequence against Arthrobacter sp. StoSoilB22 DNA

For a better understanding of the function pKARA_RT3, we analyzed the amino acid sequence with UniProt BLAST analysis. The results showed with 93.2% identity to be styrene monooxygenase StyA putative substrate binding domain-containing protein (primary accesion A0A4Y3NB75). This result was consistent with the experimental data of the activity of pKARA_RT3 to metabolize indigo to indole compounds.

Figure 2: Dot plot of nucleotide sequence of pKARA_RT3 against nucleotide sequence of Styrene monooxygenase.

For further verification of pKARA_RT3 structure, we employed NCBI Domain architecture search. The two structures this tool identified were NAD-binding domain and a Fe-S cluster, as well Styrene monooxygenase A putative substrate binding domain. The results obtained by three different analysis were consistent and we were able to confirm the origin and function of the pKARA_RT3 protein.

References

1. O’Connor, K.E., Dobson, A.D. and Hartmans, S. (1997) ‘Indigo formation by microorganisms expressing styrene monooxygenase activity’, Applied and Environmental Microbiology, 63(11), pp. 4287–4291. doi:10.1128/aem.63.11.4287-4291.1997. Sequence and Features BBa_K4719009 SequenceAndFeatures

Functional Parameters

BBa_K4719009 parameters