Team:HSiTAIWAN/Project/Assay/Copper
Oh hello there,I am Charlie,a trusty member of the "Herb Tasters" and also the brainiest E. coli in the colony.
I know all the secrets of Chinese herbs and their magical healing powers.
If you are up to a challenge,find me at team HSiTW at the jamboree.
I am the one in a straw hat,showing them pearls.I will be waiting.
Muhahahahaha
Hi there! My name is Nu Zhen Chi. This is how I look like.
Take a closer look; guess which part of me is used as medicine?
(1) the root 
(2) the stem
(3) the leaf
(4) the seed
Ans.(4) the seed
Name: 女貞子 (Nu Zhen Chi)
Botanical Name: Ligustrum lucidum Aiton
I can treat people who are yin deficient, and liver problems that cause dizziness,cataract of the eyes,
lower back pain, premature graying of the hair and tinnitus.
Hello! My name is Chuan Xiong. This is how I look like.
Make a guess, which part of me is used as medicine?
(1) the root 
(2) the stem
(3) the leaf
(4) the seed
Ans.(1) the root
Name: 川芎 (Chuan Xiong)
Botanical Name: Ligusticum chuanxiong Hort
I help with blood regulation to prevent relevant to blood stasis and non-stop bleeding.I can also strengthen your qi circulation.
In addition, I relieve you of physical pain,
such as headaches, abdominal aches, chest pain, and muscle pain.
Finally, I free the ladies of menstrual disorders and amenorrhea.
What’s up? My name is Dang Gui. I can:
(1) stop coughing 
(2) regulate mense
(3) reduce internal heat
Ans.(2) Regulate mense
Name: 當歸 (Dang Gui)
Botanical Name: Angelica sinensis (Oliv.) Diels
I can remove blood stasis and clots, so I am usually used to regulate menses,lubricate intestines to correct constipation, reduce swelling, expel pus.
Reference
臧堃堂 (2005) 中華材輕百科-現代版本草綱目，山岳文化出版社，台北
Non-Profit Organization Brion Research Institute of Taiwan.
Chinese Herb Gallery. Jade Institute
Herbal Glossary. Shen-Nong- Chinese Traditional Medicine
Acknowledgement
Thank you for Non-Profit Organization Brion Resaerch Institute of Taiwan that provide us Chinese herbs and photos.
Cu2+ Assay
- Functional Assay
- Chinese Herb Extract
- Chelating Agent
Experiment Procedures, Results, Discussion
Functional Assay "Click here to see Arsenic"
The main purposes of our functional testing are determining whether our bacteria can survive toxic environments and whether they can display fluorescent characteristics. In order to examine the survival of the bacteria, we first measure the optical density (600nm) of the bacterial fluid every half an hour. Then to prevent the deviation of initial bacteria count, we organize the results by calculating the doubling time of the bacteria. Next, we measure the fluorescence intensity of bacteria at an excitation wavelength of 470~485nm and an emission wavelength of 508~538nm for every half an hour.
Growth Curve
Fluorescence Intensity
Discussion
From graph 1, we can see that the growth rate of bacteria in concentrations of 3 ppm to 3*10-10 ppm between 3 to 5 hours does not differ. (We have done an initial data processing by taking logarithms during the bacteria’s exponential phase) That is to say, that the survival rate, reproductive rate of our bacteria is not interfered. However, the growth rate of our positive control appears to be lower than the other samples. We are guessing it is because the strong green fluorescence disturbs the wave absorption at the wavelength of 600nm, which is commonly used to measure the optical density ofbacteria. Keep in mind that for the purpose of easy labeling, the “positive control” here is not a genuine control of our testing of optical density, it is the positive control for fluorescence intensity testing (k608010). So, the true positive control for testing growth rates should be the “control”, which is the sample that contains no copper ion in its environment. Also from the same graph, we can see that the bacteria manipulate every hour and are especially significant after 5 hours.
From graph 2, we can examine that the growth of fluorescence intensity over the past 8 hours. The fluorescence expression rises rapidly after culturing for 5 hours, which matches perfectly that the fluorescence intensity does have a positive correlation with the amount of susceptible bacteria. Withal, we cannot be sure if it is the bacteria amount or the fluorescence itself that grows overtime that causes the expression to enhance. We are willing to do a further investigation. Since we have analyzed from the optical density testing, we know that the concentration of 3 ppm of copper ion does not affect the growth rate of E. coli and has the best expression. Yet, we hope to figure out the detection limit and might increase the concentration of copper ions. We are looking forward to find a more suitable, high fluorescent expression, concentration level.
Graph 1. The optical density by log2 of E. coli in its exponential phase.
Graph 2. The fluorescence intensity of E. coli for 8 hours.
Result
Different concentration levels of copper ions will not defile the growth of our bacteria. Our bacteria’s fluorescence intensity can measure the copper concentration in quantities.
Chinese Herb Extract "Click here to see Arsenic"
We want to test the function of our bacteria in Chinese Medicine. First, we conducted our experiment with herbal extract. Herbal extract is basically the production of raw herbs brewed together in the condition of high temperature and low pressure. The process can eliminate excess substances and other microorganisms. Thus, it will be a more suitable experiment subject. This year, we chose 9 types of the most common Chinese Medicine extract. We measured the growth curve, doubling time, fluorescence intensity of the bacteria cultured inside these extract fluid.
We noticed that some of the Chinese Medicine might absorb toxic ions. Hence, we manipulated a chelating agent to ligand the ions. We later ran a few experiments to ensure whether the chelating agent can cope with the issue of ion absorption and if the chelating agent itself can induce the fluorescent expression of our bacteria.
Growth Curve
Fluorescence Intensity
Discussion
Although we manipulated a few slight changes between data, but we can still acknowledge an abstract of different Chinese Medicine. Aside from Scutellaria, Bupleurum, and Ligusticum (last two not shown), the other 6 Chinese Medicine appears to be auspicious to the growth curve and fluorescence intensity of the E. coli. With our estimation, we guess that our E. coli can endure a bulk proportion of Chinese Herb; as for the few herbs that may stifle the results, we hope to create a data base for people to easily correspond to different herbs and know our product’s detection limit. Fortunately, we stumbled across a paper claiming that Scutellaria absorbs copper ions by its very own nature. Hence, it perfectly makes sense to the declining results of fluorescence overtime. To cope with this situation, we decide to add chelating agents into our product to prevent the absorption. Further experiments are expected to examine whether the agent will or will not interfere the bacteria, and the efficiency of different material agents. Lastly, we find that copper ions at the concentration of 27 ppm (the law standard in Taiwan is 20 ppm) may suppress the growth and survival of bacteria; yet, the remaining bacteria can express a high level of fluorescence intensity, which means they still great potential even in high toxic concentration levels.
Meanwhile, we think that the initial bacteria count may immensely influence our experiment time. Due to the fact that we want to create a rapid scanning, we might increase the bacterial density after this series of Chinese medicine experiments.
Graph 3. The optical density by log2 of E. coli in Schutellaria during its exponential phase.
Graph 4. The fluorescence intensity of E. coli in Schutellaria for 8 hours.
Result
Although the growth rate of E. coli in Schutellaria will not be interfered, the fluorescence intensity declines overtime.
Graph 5. The optical density by log2 of E. coli in Peppermint during its exponential phase.
Graph 6. The fluorescence intensity of E. coli in Peppermint for 8 hours.
Result
The bacteria can grow affectively in Peppermint and can express smoothly. Thus, our bacteria are fully functional in Peppermint.
Graph 7. The optical density by log2 of E. coli in Modified Ease Powder during its exponential phase.
Graph 8. The fluorescence intensity of E. coli in Modified Ease Powder for 8 hours.
Result
The bacteria can grow affectively in Modified Ease Powder and can express smoothly. Thus, our bacteria are fully functional inModified Ease Powder.
Graph 9. The optical density by log2 of E. coli in Licorice during its exponential phase.
Graph 10. The fluorescence intensity of E. coli in Licorice for 8 hours.
Result
The bacteria can grow affectively in Licorice and can express smoothly. Thus, our bacteria are fully functional in Licorice.
Graph 11. The optical density by log2 of E. coli in Angelica during its exponential phase.
Graph 12. The fluorescence intensity of E. coli in Angelica for 8 hours.
Result
The bacteria can grow affectively in Angelica and can express smoothly. Thus, our bacteria are fully functional in Angelica.
Graph 13. The optical density by log2 of E. coli in Ligustrum Seed during its exponential phase.
Graph 14. The fluorescence intensity of E. coli in Ligustrum Seed for 8 hours.
Result
The bacteria can grow affectively in Ligustrum Seed and can express smoothly. Thus, our bacteria are fully functional in Ligustrum Seed.
Graph 15. The optical density by log2 of E. coli in Four Agent Soup for 8 hours.
Graph 16. The fluorescence intensity of E. coli in Four Agent Soup for 8 hours.
Result
The bacteria can grow affectively in Four Agent Soup and can express smoothly. Thus, our bacteria are fully functional in Four Agent Soup.
Chelating Agent
To cope with the fact that some herbs may absorb the ions within their environment, we came up with a solution of adding a chelating agent, EDTA. EDTA is short for ethylenediaminetetraacetic acid, and is a commonly used chelating agent today. This agent is proven to be little harm to human’s health and is widely applied on medical treatment such as treating heavy metal poisoning1. Hence, we take advantage of this harmless yet efficient chelating agent and hope to ligand the ions inside the medicine.
Growth Curve
Fluorescence Intensity
Discussion
From graph 17 and graph 18, we can assume that EDTA alone will not interfere with the growth rate of our E. coli. Also, we can see that the component of EDTA combined with copper ion can still regulate the fluorescence expression of our bacteria. From bar chart 1 and bar chart 2, there is a significant sign that some Chinese Medicine, Scutellariae in this case, indeed stifles the expression of fluorescence. So, to deal with this issue, adding a chelating agent can be ideal. In this case, our chelating agent EDTA can even enhance the intensity of our bacteria. In conclusion, our chelating agent is an appropriate solution to the problem of Chinese Medicine absorption.
Graph 17. The optical density by log2 of E. coli in Schutellaria extract.
Graph 18. The optical density by log2 of E. coli in raw Schutellaria.
Bar Chart 1. Fluorescence intensity of E. coli in Schutellaria extract at second hour.
Bar Chart 2. Fluorescence intensity of E. coli in raw Schetellaria at third hour.
Result
The chelating agent can achieve ligand exchange and increase the fluorescent expression of bacteria.
