Difference between revisions of "Part:BBa K4719027"

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−	Colony PCR and restriction digestion analysis allowed us to select several promising constructs. Whole plasmid sequencing revealed pSEVA331-Bb-AGM1-GFA1-GNA1-UAP1-AnCDA did not contain any deleterious mutations and was successfully transformed into electrocompetent <i>K. xylinus</i> cells as seen in Figure 1.	+	Whole plasmid sequencing revealed pSEVA331-Bb-AGM1-GFA1-GNA1-UAP1-AnCDA did not contain any deleterious mutations and was successfully transformed into electrocompetent <i>K. xylinus</i> cells as seen in Figure 1.
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Revision as of 14:14, 8 October 2023

AGM1-GFA1-GNA1-UAP1-AnCDA operon for one-step bacterial cellulose-chitosan synthesis
Sequence and Features

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 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 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 composite of bacterial cellulose and polyhydroxybutyrate (PHB), which is synthesized by K. xylinus.

Bacterial cellulose-chitin polymer was achieved by increasing the production of UDP-N-acetylglucosamine, which can be recognized as a viable substrate for cellulose synthase and incorporated in the bacterial cellulose polymer. We employed two strategies to produce this material. The first approach was to add N-acetylglucosamine into the growth medium BBa_K4719013, and the second one was the production of N-acetylglucosamine by K. xylinus from simple sugars such as glucose, fructose, and saccharose in the growth medium BBa_K4719014. After achieving bacterial cellulose-chitin copolymer, we had to deacetylase this material to produce bacterial cellulose-chitosan copolymer BBa_K4719019, BBa_K4719020, BBa_K4719024. Our final objective was to combine the two necessary production steps for bacterial cellulose-chitosan copolymer into a single process.

Usage and Biology

This construct is a combination of BBa_K4719014, BBa_K4719025 and BBa_K4719011. Expression of this composite in K. xylinus results in a single-tube synthesis of bacterial cellulose-chitosan copolymer. The composite consists of an operon of synthesis and incorporation of N-acetylglucosamine, producing a bacterial cellulose-chitin copolymer, followed by signal peptide sequence fused to deacetylase for direct deacetylation reaction outside the bacteria. Incorporation of N-acetylglucosamine has been previously demonstrated by M. H. Tan (2019) and V. Yadav (2010), since the cellulose synthase can recognize both UDP-glucose and UDP-N-acetylglucosamine as substrates. K. xylinus requires genetic modification to incorporate GlCNAc into the bacterial cellulose as the natural conversion to UDP-GlcNAc is inefficient. The first four genes in this composite code for proteins responsible for converting various carbon sources such as glucose, fructose, and sucrose to UDP-GlcNAc. Another part of this composite is a signal peptide sequence BBa_K4719025 characteristic to K. xylinus fused to chitin deacetylase AnCDA BBa_K4719011.

Bacterial cellulose-chitosan copolymer has applications in the biomedicine field due to in vivo biodegradability by the lysosome. What is more, the bacterial cellulose-chitosan copolymer is a convenient platform for further modifications that would aid in solving the need for the promotion of tissue development. The uncovered amino groups are susceptible to enzymes catalyzing an addition of targeted organic chemistry groups. For instance, after modifying reaction conditions, deacetylase ClCDA BBa_K4719024 can propylate chitosan, which can later be used for click chemistry reactions [3]. In the future, specific targets like drugs or amino acids could be linked to the polymer, promoting the healing properties of the material [4].

Since polymer production occurs in K. xylinus it requires a specific plasmid backbone (pSEVA331-Bb) 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 in order to preserve Anderson promoter J23104 BBa_J23104, ribose binding site BBa_B0034 and terminator BBa_B0015. The construct was cloned by utilizing BBa_K4719014 as a plasmid backbone containing bacterial cellulose-chitin synthesis operon where signal peptide sequence and deacetylase AnCDA BBa_K4719011 were assembled into the backbone by Gibson assembly.

Experimental characterization

Verification and transformation of the single-tube synthesis of bacterial cellulose-chitosan copolymer composite

Whole plasmid sequencing revealed pSEVA331-Bb-AGM1-GFA1-GNA1-UAP1-AnCDA did not contain any deleterious mutations and was successfully transformed into electrocompetent K. xylinus cells as seen in Figure 1.

References

1.Teh, M.Y. et al. (2019) ‘An expanded synthetic biology toolkit for gene expression control in acetobacteraceae’, ACS Synthetic Biology, 8(4), pp. 708–723. doi:10.1021/acssynbio.8b00168.
2.Yadav, V. et al. (2010) ‘Novel in vivo degradable cellulose-chitin copolymer from metabolically engineered gluconacetobacter xylinus’, Applied and Environmental Microbiology, 76(18), pp. 6257–6265. doi:10.1128/aem.00698-10.
3.Lima, R., Fernandes, C. and Pinto, M.M. (2022) ‘Molecular modifications, biological activities, and applications of chitosan and derivatives: A recent update’, Chirality, 34(9), pp. 1166–1190. doi:10.1002/chir.23477.
4.Torkaman, S. et al. (2021) ‘Modification of chitosan using amino acids for wound healing purposes: A Review’, Carbohydrate Polymers, 258, p. 117675. doi:10.1016/j.carbpol.2021.117675.