Levan, a prevalent polysaccharide, is primarily composed of levulose. It boasts numerous advantageous properties, making it a staple in diverse industrial applications, such as in foods, cosmetics, and pharmaceuticals [1]. We're intrigued by its potential anticancer benefits. It's alarming that in 2020 alone, over 10 billion individuals succumbed to cancer [2]. While studies have highlighted the anticancer properties of levan, its accurate measurement challenges and inefficient screening methodologies have curtailed its potential for mass production and broader everyday benefits. Our project sets forth with two primary objectives: 1) enhancing the visibility of levan through fluorescence for more straightforward identification and measurement, and 2) through a series of constructed plasmids, experimentally determine which promoter optimally boosts levan's production efficiency.

Red fluorescent protein (RFP) is a versatile biological marker for monitoring physiological processes and visualizing protein localization. That flash of red is like a pilot light. Correlating fluorescence levels with levan yields not only accelerates the screening of microbial variants that produce high yields of levan, but also makes its yield "visible". The fluorescence of RFP and the red color visible to the naked eye turn colorless into color.

The poor efficiency and high cost of levan's detection and production are meaningful and urgent to be solved, due to the fact that levan is discovered with such good and wide range of use in meditation and others that it shows a massive future market prospect, which have mentioned above. So as to solve both of these issues, our team designed six plasmids. In addition, the sucrose-decomposing sucrase gene sacB was selected. Gram-positive bacteria such as sacB from Bacillus subtilis were expressed in E.coli and may be lethal ; however, our sacB gene was derived from Zymomonas mobilis, and expressed in E.coli is not lethal because there is no signal peptide. In the constructing of the first and second plasmids, we added levan gene and its controlling gene into the empty plasmid we are using: pUC19. During the construction of plasmid three and four, we added RFP gene and its controlling gene into our plasmid via homologous recombination, which after these steps, the levan fluoresced red that people can easily identify it by human eyes and also precisely by machines. In an effort to increase productivity of levan even further, we keep on constructing the fifth and sixth plasmids. We tested two different promoter s : plac and uv5 which liked the motor of levan and the results showed that the sixth plasmid exhibited an advance in productivity.

References

[1] Feng, J., Gu, Y., Quan, Y. et al. (2015). Recruiting a new strategy to improve levan production in Bacillus amyloliquefaciens. Sci Rep 5, 13814

[2] Cancer - World Health Organization(WHO). (2020) the key facts about cancer. (who.int )

[3] Yanase H , Fujimoto J , Maeda M ,et al.Expression of the Extracellular Levansucrase and Invertase Genes fromZymomonas mobilisinEscherichia coliCells[J].Bioscience, Biotechnology, and Biochemistry, 2014.DOI:10.1271/bbb.62.1802.