Team:UST Beijing/Experiments
Ginseng products offer a unique opportunity to meet the atherosclerosis challenge. Herbs containing ginsenosides include: Ginseng, Western Ginseng, Notoginseng, Jiaogulan etc. Current herb preparation and administration practice results in poor absorption profile limit its efficacy and cost-effectiveness. Since the ginseno-sterols are responsible for their main pharmacological effects, how to achieve effective concentration of sterol in the human body becomes critical.
In the past, two approaches have been tried to achieve this: (1) Synthesize ginseno-sterols in situ. Pro: no need to plant ginseng and harvest, continuous supply of ginseno-sterols; Con: interference with host physiology, lack of control in production. (2) Produce beta-glucosides in the gut micro-organism. Pro: convenient to hydrolyze ginsenosides in the gut; Con: interference with host gut physiology and probiotics.
In the current third approach, we use chemical reaction to hydrolyze the conjugated sugars, to satisfy “No-release” policy if iGEM safety requirement.
A synthetic beta-glucosidase gene is introduced into E.coli, along with PNPG as illustrated below. The enzyme (3D structure is displayed on the left) will make a yellow color product in the medium, which is measured by spectrometry
1. Thin—layer chromatography
Experimental purpose: Through parallel experiment of different developing methods, we identified a suitable condition in TLC system to separate ginsenosides.
Standarded samples: Rb1, Re1, Rg1 10mg/ml
After many preliminary experiments, we try to find out a general range of proportion. Here is the last TLC experiment.
chromogenic agent: concentrated sulfuric acid: carbinol = 1:9
standarded sample: extract ginsenosides from traditional Chinese medicine with n-butanol
Result: after change the ratio of chromogenic agent,the experimental phenomenon is much more clear for observing and measuring.
Change the developing agent ratio(see table below)
Result: it turns out when the ratio of developing agent is 10:2.5:0.25 and the ratio of chromogenic agent is 9:1,the number of Rf is relatively ideal.
2. A synthetic beta-glucosidase gene is introduced into E.coli, which is cultured along with PNPG as illustrated below. The enzyme (3D structure is displayed on the left) will make a yellow color product in the medium, which is measured by spectrometry.
Experiment assignment：
We set three different concentrations of PNPG in 2.5%, 5%, 10% and chose ten different germs (including germ 1 without plasmids) to examine their OD (optional density) by spectrometer once hour.
Specific experimental scheme：
Plate E.coli (empty, without Beta glycosidase plasmid) and transformed E.coli on solid medium and culture overnight to obtain monoclonal bacteria and repeat the latter for three times.（temperature：37℃）
Pick the monoclonal bacteria to 2ml liquid LB medium and culture overnight.（temperature：37℃）
Isolate E.coli(BL21-β) from 2 ml LB culture and grow E.coli(BL21-β) in M9 culture for four to six hours.（temperature：37℃）Repeat it for several times.
Use PNPG to verify whether E.coli is modified by Beta glycosidase plasmid.Put samples（including E.coli without Beta glycosidase plasmid) at three different concentrations of 5，10，20% in a 96-well plate by micropipet.
Use ultraviolet spectrophotometer to measure OD value every other hour for 6 times to test whether the plasmid in E. coli was expressed. The color is getting yellow visibly.
Initial data：
Time-dependent production of hydrolzyed color product by beta-glucosidase under different culture conditions:
As you can see from the above picture, the trend of data curve continues upward. That is to say, PNPG is decomposed，which confirms plasmids are positively transferred.
3. Use chemical method to hydrolyze ginsenoside.
Chemical hydrolysis of have been well studied and widely reported in the past. Methods include hydrochloric acid, sodium hydroxide, lactic acid, acidic amino acids, acetic acid, etc. The published methods could generate partial hydrolyzed ginsenosides. Strong acid hydrolyzation results in modification of sterol side chains, as exemplified in the following.
In the current approach, we used a combination of water, butanol, and acetic acid with a proprietary ratio to the ginsenoside substrate mixture to perform the hydrolysis:
Experimental procedure:
①use different hydrolysis system to hydrolyze ginsenosides.
②Separate hydrolyzed ginsenosides by standarded TLC system.
③utilize Double Gene report Test system built in Laboratory to test the bioactivity of hydrolyzed ginsenosides.
