Composite

Part:BBa_K4719014

Designed by: Auguste Stankeviciute   Group: iGEM23_Vilnius-Lithuania   (2023-09-03)

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

Sequence and Features


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

This specific composite part was used for bacterial cellulose-chitin polymer production. It 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 common sugars such as glucose, fructose, and sucrose 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), ribosome 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 copolymer 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 growth conditions for the polymer with the highest content of N-acetylglucosamine.

Figure 1. Bacterial cellulose-chitin copolymer. A - bacterial cellulose grown on 2 % glucose (control group). B - bacterial cellulose-chitin copolymer grown on 2 % fructose. C - bacterial cellulose-chitin copolymer grown on 2 % sucrose. D - bacterial cellulose-chitin copolymer grown on 2 % fructose.

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. We were unable to conduct a quantitative analysis of the results due to a shortage of time for generating biological replicates. But, 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.

Figure 2. RT-qPCR Ct values for AGM1-GFA1-GNA1-UAP1. As expected, Ct value for the housekeeping gene of DNA gyrase subunit B (gyrB) is lowest compared to the operon, indicating a higher expression. When comparing expression between genes in AGM1-GFA1-GNA1-UAP1, we noticed that Ct values are very similar for all four genes, meaning that the whole operon is transcribed.

FTIR spectra of bacterial cellulose-chitin copolymer

Figure 3. FTIR spectra of bacterial cellulose-chitin copolymer. A - natural bacterial cellulose control. B - bacterial cellulose produced by unmodified K. xylinus grown on 1% glucose and 1% N-acetylglucosamine. C - K. xylinus transformed with AGM1-NAG5-UAP1 (BBa_K4719013) producing bacterial cellulose-chitin copolymer grown on 1% glucose and 1% N-acetylglucosamine. D - K. xylinus transformed with AGM1-GFA1-GNA1-UAP1 producing bacterial cellulose-chitin copolymer grown on 2% glucose. E - K. xylinus transformed with AGM1-GFA1-GNA1-UAP1 producing bacterial cellulose-chitin copolymer grown on 2% sucrose. F - K. xylinus transformed with AGM1-GFA1-GNA1-UAP1 producing bacterial cellulose-chitin copolymer grown on 2% fructose. G - K. xylinus transformed with AGM1-GFA1-GNA1-UAP1 producing bacterial cellulose-chitin copolymer grown on 1% sucrose and 1% fructose.

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 indicate that the polymer composition has changed from natural bacterial cellulose to bacterial cellulose-chitin copolymer.

Fluorescence 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 detectable 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 copolymer. As expected, fructose is preferred because it has the most direct path for synthesis.

Figure 4. Fluorescent lectin staining with WGA. A - bacterial cellulose-chitin grown on 2 % glucose. B - bacterial cellulose grown on 2 % fructose. C - bacterial cellulose-chitin copolymer grown on 2 % sucrose. D - bacterial cellulose-chitin copolymer grown on 1 % glucose and 1 % fructose. E - bacterial cellulose-chitin copolymer grown on 1 % glucose and 1 % sucrose. F - bacterial cellulose-chitin copolymer grown on 1 % fructose and 1 % sucrose. G - bacterial cellulose control group. H - bacterial cellulose grown on 1 % glucose and 1 % N-acetylglucosamine. The best results were achieved with 2 % fructose, 1 % fructose and 1 % sucrose.

Chracterization of polymer surface with SEM

To verify bacterial cellulose-chitin polymer structural differences from natural bacterial cellulose, we performed scanning electron microscopy (SEM).

Figure 5. Bacterial cellulose-chitin copolymer surface characterization by SEM. A - bacterial cellulose grown on 2 % glucose (control group). B - bacterial cellulose-chitin copolymer grown on 2 % fructose, C - bacterial cellulose-chitin copolymer grown on 1 % fructose and 1 % sucrose. D - bacterial cellulose-chitin copolymer grown on 2 % glucose. The arrangement of fibers is finer in the copolymer due to altered hydrogen bonding properties between glucose monomers. The crystallinity decreases as the fibers of the polymer do not have such strong intramolecular bonds. The biggest difference in the structure is seen in the copolymer grown on 1 % fructose and 1 % sucrose.

Growth burden

In order to work with E. coli for designing constructs and testing synthetic biology systems, the growth burden of said synthetic biology parts has to be measured. We performed growth burden evaluation by measuring OD600 for five hours of modified and unmodified E. coli DH5α. The composite AGM1,GFA1,GNA1,UAP1 did not inhibit the growth of E. coli as seen in Figure 6.

Figure 6. growth burden of AGM1,GFA1,GNA1,UAP1 composite.

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. Socrates, G. Infrared and Raman characteristic group frequencies: tables and charts. John Wiley and Sons, LTD. - 3rd ed., 2002
4.Barth, A. (2007) ‘Infrared spectroscopy of proteins’, Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1767(9), pp. 1073–1101. doi:10.1016/j.bbabio.2007.06.004.
5.Bonacorsi, S. et al. (2021) ‘Systematic review on the correlation of quantitative PCR cycle threshold values of gastrointestinal pathogens with patient clinical presentation and outcomes’, Frontiers in Medicine, 8. doi:10.3389/fmed.2021.711809.