Difference between revisions of "Part:BBa K4719017"
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__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K4719017 short</partinfo> | <partinfo>BBa_K4719017 short</partinfo> | ||
| − | + | ==Introduction== | |
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. Firstly, we chose to produce a cellulose-chitin polymer 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 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 composite of bacterial cellulose and polyhydroxybutyrate (PHB), which is synthesized by ''K. xylinus''. | 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. Firstly, we chose to produce a cellulose-chitin polymer 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 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 composite of bacterial cellulose and polyhydroxybutyrate (PHB), which is synthesized by ''K. xylinus''. | ||
<br> | <br> | ||
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We produced bacterial cellulose - PHB composite by introducing PHB synthesis operon into ''K. xylinus'' [https://parts.igem.org/Part:BBa_K4719017 BBa_K4719017]. The bacteria simultaneously produce both polymers combined into the same material during the purification process. | We produced bacterial cellulose - PHB composite by introducing PHB synthesis operon into ''K. xylinus'' [https://parts.igem.org/Part:BBa_K4719017 BBa_K4719017]. The bacteria simultaneously produce both polymers combined into the same material during the purification process. | ||
| − | + | ==Usage and Biology== | |
This construct 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 procedure at boiling temperatures is required. | This construct 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 procedure at boiling temperatures is required. | ||
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Since polymer production occurs in ''K. xylinus'' requires a specific plasmid (pSEVA331-Bb) backbone for successful replication. We choose to use [https://parts.igem.org/Part:BBa_K1321313 BBa_K1321313] as it was characterized by iGEM14_Imperial team as the most suitable synthetic biology tool for ''Komagateibacter'' species. We performed PCR of the plasmid eliminating mRFP to preserve Anderson promoter J23104 [https://parts.igem.org/Part:BBa_J23104 BBa_J23104], RBS [https://parts.igem.org/Part:BBa_B0034 BBa_B0034] and terminator [https://parts.igem.org/Part:BBa_B0015 BBa_B0015]. ''phaC'', ''phaA'', ''phaB'' was assembled into the backbone by Gibson assembly. | Since polymer production occurs in ''K. xylinus'' requires a specific plasmid (pSEVA331-Bb) backbone for successful replication. We choose to use [https://parts.igem.org/Part:BBa_K1321313 BBa_K1321313] as it was characterized by iGEM14_Imperial team as the most suitable synthetic biology tool for ''Komagateibacter'' species. We performed PCR of the plasmid eliminating mRFP to preserve Anderson promoter J23104 [https://parts.igem.org/Part:BBa_J23104 BBa_J23104], RBS [https://parts.igem.org/Part:BBa_B0034 BBa_B0034] and terminator [https://parts.igem.org/Part:BBa_B0015 BBa_B0015]. ''phaC'', ''phaA'', ''phaB'' was assembled into the backbone by Gibson assembly. | ||
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| + | ==Experimental characterization== | ||
| + | <html> | ||
| + | <body> | ||
| + | <h3>Polymer production</h3> | ||
| + | <p> | ||
| + | Bacterial cellulose and polyhydroxybutyrate composite is synthesized by <i>K. xylinus</i> grown in the Glucose Yeast Extract broth (GYB) while shaking at 180 rpm at 28°C, for 7 days. As a carbon source, we used 2% glucose. | ||
| + | </p> | ||
| + | <figure> | ||
| + | <div class = "center" > | ||
| + | <center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/phb-vs-control.jpg" style = "width:900px;"></center> | ||
| + | </div> | ||
| + | <figcaption><center>Figure 1: A - bacterial cellulose control group grown on 2% glucose. B - bacterial cellulose-PHB composite </center></figcaption> | ||
| + | </figure> | ||
| + | </p> | ||
===Regulated PHB production in ''K. xylinus''=== | ===Regulated PHB production in ''K. xylinus''=== | ||
Revision as of 13:53, 22 September 2023
phaCAB operon for polyhydroxybutyrate synthesis in K. xylinus
Introduction
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. Firstly, we chose to produce a cellulose-chitin polymer 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 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 composite of bacterial cellulose and polyhydroxybutyrate (PHB), which is synthesized by K. xylinus.
We produced bacterial cellulose - PHB composite by introducing PHB synthesis operon into K. xylinus BBa_K4719017. The bacteria simultaneously produce both polymers combined into the same material during the purification process.
Usage and Biology
This construct 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 procedure at boiling temperatures is required.
Bacterial cellulose-PHB composite is an alternative to petroleum-based plastics. The advantage of this material is enhanced strenght and resistance, accelerated rate of biodegradation (1).
Since polymer production occurs in K. xylinus requires a specific plasmid (pSEVA331-Bb) backbone for successful replication. We choose to use BBa_K1321313 as it was characterized by iGEM14_Imperial team as the most suitable synthetic biology tool for Komagateibacter species. We performed PCR of the plasmid eliminating mRFP to preserve Anderson promoter J23104 BBa_J23104, RBS BBa_B0034 and terminator BBa_B0015. phaC, phaA, phaB was assembled into the backbone by Gibson assembly.
Experimental characterization
Polymer production
Bacterial cellulose and polyhydroxybutyrate composite is synthesized by K. xylinus grown in the Glucose Yeast Extract broth (GYB) while shaking at 180 rpm at 28°C, for 7 days. As a carbon source, we used 2% glucose.
