iGEM 2018 — Team UST_Beijing

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# Team:UST Beijing/Notebook

Team:UST Beijing - 2018.igem.org

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  + [HumanPractice](https://2018.igem.org/Team%3AUST_Beijing/Human_Practices)
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# Lab

# Notebook

# PROTOCOLS

# Preparing competent cell

1. Incubate E. coli overnight.

2. Dilute cultures in 1:100 and incubate at 37°C and 200rmp for 2-3 hours.

3. Measure Abs600 (between 0.3 and 0.5).

4. Take 3mL samples on ice for 10 min.

5. Centrifuge at 12000rmp for 2 mins and dump the liquid.

6. Add 1mL CaCl2 (0.1mol/L) into samples, resuspend and repeat the last step.

7. Add 500uL CaCl2 (0.1mol/L) into samples, resuspend and repeat the cen-trifugation.

8. Add 100uL CaCl2 (0.1mol/L) into samples, resuspend.

# Transformation

1. Take 5uL plasmid into 100uL E. coli BL21 competent cell and put it on ice for 30 min.

2. Heat shock at 42°C for 45s.

3. Add 450uL LB medium into samples incubate at 37°C and 200rmp for 45 min.

4. Take 100uL samples on LB agar medium (contain chloramphenicol) and in-cubate at 37°C.

# Veryfication

1. Pick the monoclonal bacteria to 2ml liquid LB medium (contain chloram-phenicol) and culture overnight at 37°C and 220rmp.

2. Transfer 20uL of sample to M9 medium for 4-6 hours at 37°C and 220rmp.

3. Add 100ul samples and blank control (E. coli BL21 without plasmid) into the well of 96-well plates.

4. Add three different doses of PNPG(5，10，20μL) in a 96-well plate by mi-cropipette.

5. Add ddH2O to make sure each well has 200uL in total.

![](https://static.igem.org/mediawiki/2018/6/6d/T--UST_Beijing--Web_protocol-96-wells.JPG)

6. Use ultraviolet spectrophotometer to measure Abs405 value every other hour.

# Hydrolyzing Ginsenoside

1. Add engineered strain 2mL and M9 medium 200mL (contain Chloram-phenicol) into fermentation tank.

2. Set heating equipment (household appliances which is used to make yo-gurt or wine), choose “yogurt” option.

3. Pump in sterile air and let bacteria grow 4-6 hours.

4. Add ginsenoside powder into fermentation tank directly.

# Thin—layer chromatography

1. Prepare Developing solvents 5 ml （CHCl3:CH3OH:H2O=10:2.5:0.5）.

2. Prepare color developing reagent（10%H2SO4） 100mL.

3. Prepare standard sample.

4. Use water saturated n-butyl alcohol to extract hydrolysate.

5. Use capillary tube to point samples onto silicon-glass plate.

![](https://static.igem.org/mediawiki/2018/0/01/T--UST_Beijing--Thinlay-1.png)
![](https://static.igem.org/mediawiki/2018/f/fb/T--UST_Beijing--Thinlay-2.png)

6. Put plate into developing solvents, and take out plate when the solvents ar-rive end line.

7. After drying, put plate into color developing reagent and take out immedi-ately.

8. Use hair dryer to accelerate the process of desiccation.

# Labnote

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# Team:UST Beijing/Experiments

Team:UST\_Beijing/Experiments

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# Experiments

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### Experimental Design

### Ginseng products offer a unique opportunity to meet the atherosclerosis challenge. Herbs containing ginsenosides include: Ginseng, Western Ginseng, Notoginseng, Jiaogulan etc. Common herb preparation and oral administration practice results in poor absorption profile thus limits 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. Our long-term goal is to improve the health-promoting effects of ginsenosides. We believe that sterols (triterpenes)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.

### 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 host gut micro-organism. Pro: convenient to hydrolyze ginsenosides in the gut; Con: interference with gut physiology and probiotics.

![](https://static.igem.org/mediawiki/2018/0/05/T--UST_Beijing--ep13.png)

### In the current third approach, we use chemical reaction to hydrolyze the conjugated sugars, to satisfy “No-release” policy if iGEM safety requirement.

![](https://static.igem.org/mediawiki/2018/3/32/T--UST_Beijing--ep14.png)

![](https://static.igem.org/mediawiki/2018/1/1c/T--UST_Beijing--ep15.png)

A synthetic beta-glucosidase gene is introduced into E.coli, which is cultured along with PNPG as illustrated below. The enzyme (structure as illustrated) will produce a yellow color product which is secreted to the medium, and 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 a single clone of bacteria and repeat the experiment for three times.（temperature：37℃）
Pick a single clone bacteria into 2ml liquid LB medium and culture overnight.（temperature：37℃）
Isolate E.coli(BL21) from 2 ml LB culture and grow E.coli(BL21) in M9 culture for four to six hours.（temperature：37℃）Repeat it for several times.
Use PNPG to verify whether E.coli is transformed by beta-glucosidase expressing plasmid. Put samples（including untransformed E.coli as negative control) at three different concentrations 5，10，20% in a 96-well plate by micropipet.
Use spectrophotometer to measure OD405nm and OD620nm value every 2 hours hour for 12 hours 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. (The blue line: OD405nm, red line: OD620nm, green line: logistic model simulation.)

![](https://static.igem.org/mediawiki/2018/4/4c/T--UST_Beijing--ep20.png)

### Use chemical methods to hydrolyze ginsenoside:

### Chemical hydrolysis of ginsenosides 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. However, strong acid hydrolyzation results in modification of sterol side chains, as examplified in the following reaction:

![](https://static.igem.org/mediawiki/2018/d/d8/T--UST_Beijing--ep001.png)

### In the current approach, we applied a combination of water, butanol, and acetic acid in a proprietary ratio to the ginsenoside substrate mixture to perform the hydrolysis:

![](https://static.igem.org/mediawiki/2018/3/3f/T--UST_Beijing--ep002.png)

### 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:

![](https://static.igem.org/mediawiki/2018/2/21/T--UST_Beijing--reporter.jpg)

### Conclusion:

### Through the chart, we can see that the hydrolyzed ginsenosides has better LXR tranactivation ability compared to the unhydrolyzed. Meanwhile, the effect of hydrolyzed ginsenosides is relatively closed to a positive control sample. To conclude, we demonstrate that our "natural RE-lease" approach is working.

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# Team:UST Beijing/Results

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TEAM

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PROJECT

[Description](https://2018.igem.org/Team%3AUST_Beijing/Description)
[Design](https://2018.igem.org/Team%3AUST_Beijing/Design)
[Experiments](https://2018.igem.org/Team%3AUST_Beijing/Experiments)
[Notebook](https://2018.igem.org/Team%3AUST_Beijing/Notebook)
[InterLab](https://2018.igem.org/Team%3AUST_Beijing/InterLab)
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PARTS

[Parts Overview](https://2018.igem.org/Team%3AUST_Beijing/Parts)
[Basic Parts](https://2018.igem.org/Team%3AUST_Beijing/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AUST_Beijing/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AUST_Beijing/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AUST_Beijing/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AUST_Beijing/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AUST_Beijing/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AUST_Beijing/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AUST_Beijing/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AUST_Beijing/Hardware)
[Measurement](https://2018.igem.org/Team%3AUST_Beijing/Measurement)
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[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=UST_Beijing)

# Results

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember all measurement and characterization data must be on part pages in the Registry.
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)