Team:UST Beijing/Model
Description
β-galactosidase is used to deglycosylate saponin of notoginseng. Our Lab have a PET-28a plasmid withβ-galactosidase gene and LacI gene. The transcription of β-galactosidase is repressed by LacI protein. But lactose and IPTG can induce the expression of LacI protein. We used a 3L fermentation tank to conduct preliminary experiments, then the enzyme was extracted from bacteria solution using glycine buffer.
Why we choose our project
As Chinese traditional medicinal materials, notoginseng has been widely recognized on its efficacy by Chinese people during thousands of years. With the developing of modern medicine, other characteristics of notoginseng have been utilizing. Now it’s been proved that notoginsenoside has therapeutic effect on hyperlipidemia and cardiovascular diseases. However, the bioavailability of saponin of notoginseng in human body can reach only 4%. Based on this premise, we hope to hydrolyze glycosyl on saponin of notoginseng molecule out of body, so that deglycosylated saponin of notoginseng can be easy for human to absorb.
Find new solid medium
Acting as solid medium for E.coli fermentation, notoginseng are boiled in water. E.coli can obtaining nutrient from notoginseng solid medium. After reaching a certain cell concentration, E.coli will express objective protein, and then saponin of notoginseng will be deglycosylated. To provide enough nutrition for the growth of E.coli, Notoginseng solid medium are anaerobically fermented by rhizopus and yeast at room temperature so that polysaccharide can be hydrolyzed to glucose, galactose, and arabinose etc. We kept in track the concentration of reducing sugar in notoginseng solid medium. The concentration of reducing sugar reached 10g/L when the E.coli fermentation began.
Double plasmids system
(CORE EXPERIMENT)
β-galactosidase is used to deglycosylate saponin of notoginseng. Our Lab have a PET-28a plasmid withβ-galactosidase gene and LacI gene. The transcription of β-galactosidase is repressed by LacI protein. But lactose and IPTG can induce the expression of LacI protein. We used a 3L fermentation tank to conduct preliminary experiments, then the enzyme was extracted from bacteria solution using glycine buffer. The result showed us that extracted solution has strong ability to hydrolyze glycosyl. However, there’s no lactose in notoginseng solid medium. In order to reduce costs, another plasmid psb1C3 which contains T7 RNA Polymerase gene and was transformed into E.coli. Psb1C3 contains T7 RNA Polymerase gene and can be regulated by pBAD. This double-plasmid system is expected to be regulated by pPAD, and expresses a large number of T7RNA polymerase to inhibit the effect of LacI repression, switch on the expression ofβ-galactosidase. It’s been reported in bibliography that the cellwall of notoginseng contains a certain concentration of arabinose. Our ultimate goal is using notoginseng to provide nutrients for E.coli in a solid state fermentation jar, E.coli can deglycosylate saponin of notoginseng as well.
Bi-induction experiments
We used lactose and arabinose as Inducer, PNPG as substrate of the enzyme to conduct bi-induction experiments. When PNPG lose its glucose, the remaining part, p-Nitrophenol would present yellow. Then the A450 of bacteria solution was measured by microplate reader. The value of A450 is proportional to enzyme activity. Contrary to expectation, enzyme activity was inversely proportional to lactose and arabinose concentration, and it’s been proved by repeated trials.We use product inhibition to explain this phenomenon. That is because breakdown products of PNPG contain a lot of monosaccharide which may inhibit hydrolysis reaction of β-galactosidase.
1.Animals experiment:To verify the effect of notoginseng, we used high-fat feeding to feed hamsters. Hypolipidemic capacity of notoginseng can be displayed by cholesterol concentration of hamsters’ serum.
2. Synthetic plasmid: Using PSB1C3 as the backbone of plasmid, we compiled the pBAD and β-galactosidase genes as the iGEM parts which need to be submitted to iGEM competition. We hope this part can combine abilities of two former parts.
Reference
1. Robert Schleif, AraC protein, regulation of the L-arabinose operon in Escherichia coli, and the light switch mechanismof AraCaction,FEMS Microbiol, Rev 34 (2010) 779–796. 2. Xin Zhang, Robert Schleif, Catabolite Gene Activator Protein Mutations Affecting Activity of the araBAD Promoter, Jounal of Bacteriology,Jan.1998, p.195–200 3. Judith A. Megerle, Georg Fritz,y Ulrich Gerland,y Kirsten Jung,z and eta, Timing and Dynamics of Single Cell Gene Expression in the Arabinose Utilization System, Biophysical Journal, Volume 95 August 2008, 2103–2115. 4. Jarno Meenakshisundaram Kandhavelu1, Samuel M. D. Oliveira1, Jerome G. Chandraseelan1, Jason Lloyd-Price1, Juha Peltonen1, Olli Yli-Harja1,Andre S. Ribeiro1, In vivo single-molecule kinetics of activation and subsequent activity of the arabinose promoter, Nucleic Acids Research, 2013, Vol. 41, No. 13, 6544–6552. 5. Casonya M. Jognson and Robert F. Schleif, In Vivo Induction Kinetics of the Arabinose Promoters in Escherichia coli, Biophysical Journal, Journal of Bacteriology, June 1995, p.3438–3442. 6. pET-28a-c(+) Vectors map. 7. M. Rossi and eta, Characterization of the plasmid pMB1 from Bifidobacterium iongum and its use for shuttle vector construction, Institut Pasteur, 1996, 147, 133-143. 8. William and Mary, An analytical model of the effect of plasmid copy number on transcriptional noise strength, iGEM 2015. 9. Jingcheng Xiao, Huimin Chen, Dian Kang, Yuhao Shao, Boyu Shen, Xinuo Li, Xiaoxi Yin, Zhangpei Zhu, Haofeng Li, Tai Rao, Lin Xie, Guangji Wang, Yan Liang, Qualitatively and Quantitatively Investigating the Regulation of Intestinal Microbiota on the Metabolism of Panax notoginseng saponins, Journal of Ethnopharmacology. 10. Du Wenxia,Duan Shufang,Yu Xiaoling,Yin Liming, Panax notoginseng saponins suppress radiation-inducedoste oporosis by regulating bone formation and resorption, Phytomedicine, 22(2015)813–819. 11. Jing-Rong Wang, Lee-Fong Yau, Wei-Na Gao, Yong Liu, Pui-Wing Yick, Liang Liu, Zhi-Hong Jiang, Quantitative Comparison and Metabolite Profiling of Saponins in Different Parts of the Root of Panax notoginseng, Journal of Agricultural and Food Chemistry. 12. Li Juan and eta, Structure and biological action on cardiovascularsystems of saponins from Panax notoginseng, China Journal of Chinese Materia Medica, Vol.40, No.17 September, 2015. 13. Trygve Brautaset and eta, Positively regulated bacterial expression systems, Microbial Biotechnology (2009) 2(1), 15–30.
