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Team:UST_Beijing/Experiments

Abstract:

Our long-term goal is to improve the health-promoting effects of ginsenosides. We believe that sterols in the ginsenosides are responsible for their main benefits.
Therefore in the past projects we engineered synthetic squalene cyclase for in situ production of ginseng-sterols in human cells and produced synthetic β-glucosidase in E.coli for removal of sugar from ginsenosides.
In the current strategy, in the wake of “No release” policy of the iGEM community, we are able to by-pass synthetic biology methods to achieve our goal by applying in vitro chemical reactions.

Ginseng products offer unique opportunity to meet the atherosclerosis challenge. Herb catalogs: 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 become 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 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 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.

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.

Time-dependent production of hydrolzyed color product by beta-glucosidase under different culture conditions:

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.

Cellular assay of Ginsenoside hydrolysate biological activity:

Discussion:

Through the chart, we can see that the hydrolyzed ginsenosides has better LXR expression intensity compared to unhydrolyzed LXR expression intensity as density arises. Meanwhile, the effect of hydrolyzed ginsenosides is relatively closed to positive contrast. That is to say, we demonstrate that our natural-re-lease’s method is valid.

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