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AGM1-GFA1-GNA1-UAP1 operon for synthesis of bacterial cellulose-chitin copolymer

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 of bacterial cellulose and polyhydroxybutyrate (PHB) composite, 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:
1. The first approach was to add N-acetylglucosamine into the growth medium BBa_K4719013.
2. 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.

Usage and Biology

This construct produces an excess of UDP-N-Acetylglucosamine (GlcNAc) in K. xylinus, which can be used by cellulose synthase to produce bacterial cellulose-chitin polymer. Incorporation of N-acetylglucosamine has been previously demonstrated by M. H. Tan (2019) and V. Yadav (2010). K. xylinus requires genetic modification in order to incorporate GlCNAc into the bacterial cellulose as the natural conversion to UDP-GlcNAc is not efficient. The genes in this composite code for proteins that are responsible for the conversion of various carbon sources such as glucose, fructose, and sucrose to UDP-GlcNAc. Therefore, the growth medium doesn't have to be supplemented with GlcNAc, this significantly reduces the cost of production.

Bacterial cellulose-chitin copolymer has applications in the biomedicine field due to in vivo biodegradability by the lysosome. However, this copolymer is an intermediate step in your biological system since we chose to improve the qualities of the material by deacetylation. This second enzymatic step produces a bacterial cellulose-chitosan copolymer that has the added benefits of antibacterial activity and amino groups for easier functionalization.

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. AGM1, GFA1, GNA1 and UAP1 were assembled into the backbone by Gibson assembly.

Experimental characterization

Polymer production

Bacterial cellulose-chitin polymer is synthesized by K. xylinus grown in the Glucose Yeast Extract broth (GYB) while shaking at 180 rpm at 28°C, for 7 days. Varying concentrations of glucose, fructose and sucrose have been added to determine the best combination of growing conditions for the polymer with the highest content of N-acetylglucosamine.

Gene expression analysis by RT-qPCR

In order to verify synthetic operon's AGM1-GFA1-GNA1-UAP1 expression in K. xylinus, we performed real-time quantitative PCR. Although we could not execute quantitative analysis of the results because we did not have enough time for biological replicates, we could compare cycle threshold (Ct) values. This measure shows how many PCR cycles are required to obtain positive results (Bonacorsi et al., 2021). Expression of every selected gene was detectable because all Ct values were lower than 37.

FTIR spectra of bacterial cellulose-chitin copolymer

The adsorption band at 1589 cm^-1 closely corresponds with OH deformation vibration as described in the literature [3]. This vibration could be attributed to 2’ OH and is the most apparent in the A curve of control bacterial cellulose polymer. The absorption intensity of this peak decreases with the incorporation of N-acetylgluscosamine. The second band at 1651 cm^-1 is amine I vibration, confirming the incorporation of N-acetylglucosamine [4]. However, the wavenumbers of bands of OH deformation vibration and amine I vibration fall into the same region therefore, the 1651 cm^-1 band in the control group of bacterial cellulose is visible. With this in mind, we can compare the compositions of polymers by observing how both bands change respectively. Since FTIR is a qualitative measure, curves show that the polymer composition has changed from natural bacterial cellulose to bacterial cellulose-chitin copolymer.

Floresence analysis of N-acetylglucosamine incorporation

To determine if the polymer contains N-acetylglucosamine residues, we used fluorescence lectin staining with wheat germ agglutinin (WGA), Alexa Fluor™ 680 conjugate selectively binding to N-acetylglucosamine. Therefore, natural cellulose should not give a visible signal at this wavelength. Construct for synthesis and incorporation of N-Acetylglucosamine into bacterial cellulose improved the fluorescence signal substantially, showing consistent distribution of the target monomers. We tested which sugars are optimal for the metabolism pathway that we engineered and resulted in the highest content of N-Acetylglucosamine in the composite. As expected, fructose is preferred because it has the most direct path for synthesis.