Effects of Green Tea On E. coli Exposed To UV Light
Rebecca Gurba
Undergraduate Student at Wofford College
Proposed Budget: $34
3rd Revision (March 3, 2002)
PROJECT SUMMARY
The question being addressed is whether green tea extract increases survival of Escherichiacoli exposed to ultraviolet radiation (UV rays).
Before the experiment is conducted, two pieces of information should be obtained.
A dilution yielding a countable number of bacteria should be established. With that dilution number, a survivorship curve experiment must be conducted to establish the optimal exposure time of bacteria to UV light to kill off 90%. An aqueous solution of green tea extract (GTE) will be created as well. The GTE of varying dilutions will be mixed with E. coli in a nutrient broth (the experimental group). The bacteria will incubate in their respective GTE nutrient broth solutions allowing for uptake of green tea extract. Small quantities will be pipetted out onto plates and exposed to UV light. The bacteria will then be incubated and their colonies will be counted and compared with controls.
It is anticipated that there will be a correlation between the number of surviving bacteria after UV exposure, and the concentration of green tea extract the bacteria were incubated.
JUSTIFICATION
The purpose of the experiment is to determine whether green tea extract increases survival rates in E. coli bacteria exposed to UV light. UV rays cause the formation of cross-linking of adjacent pyrimidine bases thereby forming pyrimadine dimers (1). These pyrimadine dimers are detrimental to the cell because they halt transcription that will inevitably lead to cell death (10). Some strains of E. coli have DNA repair enzymes which function in the reversal of dimer formations (1). The strain chosen for the experiment (B/r WP2 trp-) is one such strain. The DNA of this bacteria strain contains a DNA excise repair mechanism, similar to that of human DNA, only involving fewer genes (2). It has been suggested that it is this repair mechanism in E. coli that works in association with tannic acid and offers an enhanced protection against the mutagenic properties of UV rays (3). Green tea, along with other herbal products contains polyphenols, which impart antioxidant and anitmutagenic benefits in animals, including humans (7). Should green tea prove to offer some resistance to UV rays in E. coli, it may offer some protection in humans as well. The author speculates that daily ingestion of green tea may possibly act in a preventative manner against certain skin cancers caused by sun exposure.
BACKGROUND
Tannins are found within many species of plants such as oak gulls, hemlock, chestnut and mangroves. Tannic acid, a form of tannin, has a wide variety of medicinal uses that can be traced back to the Middle Ages (11). Not only is Tannic acid particularly effective in treating burns; it is also used to treat hemorrhoids, dysentery, conjunctivitis, and various skin diseases (11). Evidence has shown that tannic acid has antimutagenic and anti-oxidant properties (3). Due to its structure, tannic acid is said to have the ability to mediate DNA cleavage (5). Tannic acid stimulates the excision repair process of E. coli thereby removing pyrimidine dimers and allowing for synthesis of a new DNA strand (4,10). Researchers Inoue and Kuroda (4) have stated that substances showing antimutagenic properties in bacteria also show antimutagenicity in mammalian cells. This study is to see if the tannic acid contained in green tea may enhance inhibition of UV induced fatality in E. coli.
METHODS
Bacteria will be grown in a flask with nutrient broth overnight. Serial dilutions will be made to establish a countable dilution number see, Table 2. A survivorship curve will then be made by exposing E. coli of a dilution established previously to 0, 4, 8, 12, 16 and 20 minutes of UV light. This is done to determine the optimal time frame for exposure of UV to kill 90% of the bacteria.
Solutions of varying concentrations of green tea will be manufactured by percolating 4 liters of hot water through 20 grams of tea leaves making a 5% GTE solution. From this solution; a 50%, 25%,10% and 1% dilution of GTE will and mixed with 1 loopful of E. coli (strain B/r WP2 trp-) and nutrient broth. The following table provides the breakdown of samples:
Table 1: three speard plates per column
[ tea extract] / none / 50% / 25% / 10% / 1%UV / Yes / 3 / 3 / 3 / 3 / 3
No / 3 / 3 / 3 / 3 / 3
The bacteria will be incubated for 20 minutes in the appropriate GTE (9). The controls will be a loop full of E. coli in nutrient broth with specified GTE concentration with no UV exposure. A 5 ml sample will be aliquot from each solution and transferred to a spread plate. Each plate will be placed under UV lamp ( 254 nm), with cover off, 30 cm away for time obtained from survivorship curve (2, 8).
Table 2: the procedure for serial dilution
After all samples are transferred to nutrient agar plate, they will be incubated for 24 hours in 32 degree Celsius (9). Colonies per ml of sample will then be calculated by using the formula: number of colonies on plate * reciprocal of dilution of sample = number of bacteria / ml (9).
COMPLICATIONS
A major complication in this study would be the complete annihilation of the bacteria after being exposed to the UV rays. The reverse could happen where bacteria were unaffected by the UV light. Another problem would be if the amount of tannic acid in green tea were not enough to make any difference in the bacteria surviving. Green Tea extract may also work to inhibit the growth of E. coli, giving falsely negative results.
GOALS
The goals of this experiment are to see a clear difference in the amount of colonies that survived the UV rays that were also grown with the green tea extractions, versus the colonies that were not grown in green tea extract. The expectation is to demonstrate that green tea increases survival rate of E. coli from UV rays.
BUDGET JUSTIFICATION:
Items needed-
E. coli (B/r WP2 trp-) - $180.00
½ lb of green tea leafs -$3.00
Bacteria Nutrient Agar
Bacteria Nutrient Broth
Items we have-
- UV lamp- UVP model UVG-11, Mineralight Lamp, short wave UV-254 nm,115V, 60Hz, .16A
Micropipettes
Nutrient agar
Sterile plates
Inoculating loops
Incubators
REFERENCES
1. Boyer R. 1999. Concepts in Biochemistry. Brooks/ Cole Publishing Company. 330 p.
2. Vasilieva. 2001. Para-aminobenzoic acid inhibits a set of SOS functions in Escherichia coli K12. Mutation Research 496: 89-95.
3. Ahmad A, Hadi S, Khan N. 2000. Anti-oxidant, pro-oxidant properties of tannic acid and its binding to DNA. Chemico-Biological Interactions 125: 177-189.
4. Shimoi K, Kada T, Nakamura Y, Tomita L. Bio-antimutagenic effects of tannic acid on UV and chemically induced mutagenesis in Escherichia coli B/r.
5. Kuroda Y, Inoue T. 1988. Antimutagenesis by factors affecting DNA repair in bacteria. Mutation Research 202: 387-391
6. Laboratory of Health Science, Shizuoka College of Pharmaceutical Sciences, Shizuoka 422, Japan.
7.Katiyar S, Bergamo B, Vyalil P, Elmets C. 2001. Green tea polyphenols: DNA photodamage and photoimmunology. Journal of Photochemistry and Photobiology B: Biology. 2001; 65 (109-114).
8.Delpech R. 2001. Using Vibria natriegens for studying bacterial population growth, artificial selection, and the effecs of UV radiation and photo-reactiviation. Journal of Biological Education. 35: 93-97.
9.Case C, Funke B, Tortora G. 1998. Microbiology An Introduction. 6th ed. The Benjamin/ Cummings Publishing Company, Inc. 172 p.
10.Freifelder D, Malacinski G. 1998. Essentials of Molecular Biology. 3rd ed. Boston: Jones and Bartlett Publishers. P 237-239.
11. Moore M. The American Materia Medica, Therapeutics and Pharmacognosy. 2001. Home page. < Accessed 2002 February 15.