Difference between revisions of "Part:BBa K759004"
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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>. <br>
 
<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>. <br>
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Bacterial cellulose-PHB composite was achieved by introducing PHB synthesis operon into <i> K. xylinus</i>. Bacteria are able to simultaneously produce both polymers and combine them into one composite cellulose-PHB polymer.
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Bacterial cellulose-PHB composite was achieved by introducing PHB synthesis operon into <i> K. xylinus</i>. This way, bacteria are able to simultaneously produce both polymers and combine them into one composite cellulose-PHB polymer.
  
  
 
<h3>Usage and biology</h3>
 
<h3>Usage and biology</h3>
 
<p>
 
<p>
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We have used this part in the polyhydroxybutyrate (PHB) synthesis operon, which gene expression was characterized in <K. xylinus</i>. The construct <a href="https://parts.igem.org/Part:BBa_K4719017">BBa_K4719017</a> containing <i>phaA</i> is a polyhydroxybutyrate synthesis operon (<i>phaC, phaA, phaB</i>) producing PHB along with bacterial cellulose in <i>K. xylinus</i>. PHB is stored in bacteria intercellularly while cellulose is secreted outside of the cell. To combine both of these polymers washing procedure at boiling temperatures is required.
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This part was used in the polyhydroxybutyrate (PHB) operon, which gene expression was realized in <K. xylinus</i>. The construct <a href="https://parts.igem.org/Part:BBa_K4719017">BBa_K4719017</a>, containing <i>phaA</i>, is a polyhydroxybutyrate synthesis operon (<i>phaC, phaA, phaB</i>) producing PHB along with bacterial cellulose in <i>K. xylinus</i>. PHB is stored in bacteria intercellularly while cellulose is secreted outside of the cell. To combine both of these polymers washing procedures at boiling temperatures are required.
 
<br>
 
<br>
 
<br>
 
<br>
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Bacterial cellulose-PHB composite is an alternative to petroleum-based plastics. The advantage of this material is enhanced strength and resistance, accelerated rate of biodegradation [1]. </p>
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Bacterial cellulose-PHB composite is an alternative to petroleum-based plastics. The advantage of this material is enhanced strength, resistance and accelerated rate of biodegradation [1]. </p>
  
  
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<h3>Constitutive expression of PHB synthesis genes in <i>K. xylinus</i></h3>
 
<h3>Constitutive expression of PHB synthesis genes in <i>K. xylinus</i></h3>
 
<p>
 
<p>
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We have evaluated whether the <i>phaA</i> gene can by expressed in <i>K.xylinus</i> by designing PHB synthesis operon. To verify the synthesis of PHB, we supplemented growth media with 2.5&micro;l/ml Nile red A. Nile red A is used to determine the presence of PHB with fluorescence. <b>Colonies containing working constitutive PHB synthesis construct should appear red under UV light.</b>
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We have evaluated whether the <i>phaA</i> gene can by expressed in <i>K.xylinus</i> by designing PHB synthesis operon. To verify the synthesis of PHB, we supplemented growth media with 2.5&micro;l/ml Nile red A. Nile red A is used to determine the presence of PHB with fluorescence.<b>Colonies containing constitutive PHB synthesis construct should appear red under UV light.</b>
 
</p>
 
</p>
  

Revision as of 21:50, 9 October 2023

phaA (beta-ketothiolase orgin from Ralstonia eutropha)

This gene codes the enzyme“Beta- ketothiolase (acetyl-CoA acetyltransferase)”which catalyzes the conversion of two acetyl-CoA molecules into acetoacetyl-CoA. It is an enzyme that catalyzes the first step of P3(HB) producing pathway like the picture below.

This gene was cloned from pGEM phbCAB which carries the synthetic operon genes (phaA,phaB,phaC) of Ralstonia eutropha. In the pGEM phbCAB phaA, we found a mutation in the 00th base according to the data base of NCBI.


Contribution by Vilnius-Lithuania 2023

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.
Bacterial cellulose-PHB composite was achieved by introducing PHB synthesis operon into K. xylinus. This way, bacteria are able to simultaneously produce both polymers and combine them into one composite cellulose-PHB polymer.

Usage and biology

This part was used in the polyhydroxybutyrate (PHB) operon, which gene expression was realized in . The construct BBa_K4719017, containing phaA, is a polyhydroxybutyrate synthesis operon (phaC, phaA, phaB) producing PHB along with bacterial cellulose in K. xylinus. PHB is stored in bacteria intercellularly while cellulose is secreted outside of the cell. To combine both of these polymers washing procedures at boiling temperatures are required.

Bacterial cellulose-PHB composite is an alternative to petroleum-based plastics. The advantage of this material is enhanced strength, resistance and accelerated rate of biodegradation [1].

Experimental characterization

Constitutive expression of PHB synthesis genes in K. xylinus

We have evaluated whether the phaA gene can by expressed in K.xylinus by designing PHB synthesis operon. To verify the synthesis of PHB, we supplemented growth media with 2.5µl/ml Nile red A. Nile red A is used to determine the presence of PHB with fluorescence.Colonies containing constitutive PHB synthesis construct should appear red under UV light.

Figure 1: Left - bacterial cellulose control group grown on 2 % glucose (negative control). Right - constitutive gene expression construct producing bacterial cellulose-PHB composite. K. xylinus can be identified as producing PHB.

References

1. 1. Ding, R. et al. (2021) ‘The facile and controllable synthesis of a bacterial cellulose/polyhydroxybutyrate composite by co-culturing Gluconacetobacter xylinus and Ralstonia eutropha’, Carbohydrate Polymers, 252, p. 117137. doi:10.1016/j.carbpol.2020.117137.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 121
    Illegal NgoMIV site found at 217
    Illegal NgoMIV site found at 225
    Illegal NgoMIV site found at 237
    Illegal NgoMIV site found at 334
    Illegal NgoMIV site found at 574
    Illegal NgoMIV site found at 715
  • 1000
    COMPATIBLE WITH RFC[1000]