GENETICALLY ENGINEERING A NOVEL STRAIN OF

NONPATHOGENIC BACTERIA FOR PRACTICAL ARSENIC

REMOVAL

Kriti LallCastilleja School

Palo Alto, CA

INTRODUCTIONArsenic exists in mainly two forms in the environment: arsenite and arsenate. Arsenite is difficult to remove from water. Arsenate, however, is readily removed from water. Because it is so easily soluble in water, arsenite is the primary cause of arsenicosis, or arsenic poisoning caused by drinking water with high arsenic concentrations. Victims of arsenicosis suffer from diabetes, blood vessel abnormalities, cancer, and other diseases. Current methods for purifying arsenic-rich water are costly and inefficient. The purpose of this research is to investigate a novel solution to this problem: in particular, studying and manipulating a certain gene in extremophilic bacteria, known as arxA, to purify water containing arsenic.

ARSENIC: IT’S IN OUR WATERIn water, arsenic is mostly found in the form of arsenite (the toxic form that is hard to remove). Because it is so tough to take out of water (and is expensive), it is not practical for companies or organizations to remove it. And because no measures are being taken to remove it, people have no choice but to continue to drink the water with the poison.

Arsenic levels are especially high in countries like Bangladesh, India, China, and Mexico – which in turn affect the people who drink the water with the arsenic.

GLOBAL ARSENIC DISTRIBUTION

Source: WHO (World Health Organization)

ARSENICOSIS (ARSENIC POISONING)

• Arsenicosis, or arsenic poisoning, results from entry of large amounts of arsenic in the body. This cumulative buildup of toxins causes serious health problems over the long run.

• Eventually, arsenicosis leads to cancers of the liver, skin, lymphatic system, lungs, and urinary tract.

• More than 137 million people in 70 countries are currently suffering from arsenicosis, and a report recently predicted that in the next decade in Bangladesh alone, “1 out of every 10 adult deaths” will be a result of arsenicosis.

ARSENIC TODAY: EFFECTS AND REMOVAL

• Current arsenite removal methods are inefficient and cost thousands of dollars to implement. Furthermore, they often produce toxic sludge and yield little to no financial return. This is because arsenite (As3+) is non-ionic, so it’s not easily removed from water.

• Arsenate, however, (As5+) is easy to remove from water because it is ionic. Hence, it can be precipitated out of water with a compound such as ferrous sulfate.

• The problem? Most arsenic in water exists in the form of arsenite, not arsenate.

• In the summer of 2012, I learned about an extremophilic bacteria (lives in extreme environments) called MLHE-1 that has a gene called arxA, which oxidized arsenite to arsenate as part of its function. The bacteria uses this redox reaction to maintain cell growth and fix carbon.

• But the bacteria I worked with requires extreme conditions to survive. Such a bacteria would not be a good choice for practical arsenic bioremediation because of the high cost incurred to maintain a suitable environment for the bacteria itself.

• I also heard about an E. coli strain called K-12. This strain is known to be easy to work with.

• An idea was sparked…

WHAT GOT ME THINKING…

RATIONALE BEHIND THIS STUDYarxA is the gene in the bacteria strain MLHE-1 that allows the bacteria to oxidize arsenite. However, MLHE-1 is an extremophile, and requires extreme conditions to survive. Therefore, it is impractical to build a commercial or large-scale bioreactor to solve the arsenic water problem using MLHE-1. Such a bioreactor would require additional heat, nutrients, maintenance of pH, and other conditions just to keep MLHE-1 alive. This would drive up costs of the whole water purification process.

Implanting the arsenite-oxidizing gene, or arxA, in a different, commonly-found, nonpathogenic bacteria, such as E. coli strain K12, would solve the problem because the mutated bacteria would have the same arsenite-oxidizing capabilities as MLHE-1, but not require a special environment to survive.

Can E. coli strain K-12 (nonpathogenic) be

transformed to contain the gene arxA from the

extremophilic strain of bacteria, MLHE-1? If so,

will the E. coli strain successfully oxidize

arsenite to arsenate?

QUESTION

HYPOTHESIS E. coli strain K-12 can be transformed to contain the gene arxA, an arsenite oxidase. Furthermore, the new GMO will successfully oxidize arsenite to arsenate.

Strain MLHE-1

E. C

oli

stra

in K

12

• Place of Isolation: Mono Lake, CA

• Anaerobic, facultative autotrophic bacterium that respires nitrate or nitrite, and can oxidize arsenite (highly toxic and easily soluble in water) into arsenate (less toxic) using the arxA gene.

• Can grow as an anaerobic chemoautotroph by linking oxidation of arsenite, hydrogen, or sulfide to nitrate reduction

• Can perform carbon fixation during chemoautotrophic growth, but can also grow as a heterotroph on acetate with nitrate.

• Optimal growth conditions: pH 9.8, 37ºC, anaerobic environment

• Grows on Mono Lake Minimum (MLM) Medium

PRELIMINARY RESEARCH: STRAIN MLHE-1

PRELIMINARY RESEARCH: ARXA GENE

• arxA is an arsenite oxidase that enables MLHE-1 to oxidize arsenite.

• Has been identified in the genomic sequence of 4 other strains other than MLHE-1.

• Plasmid pT-BS was used during this study to transfer the arxA gene from MLHE-1 to E. coli strain K12.

• Expression: Bacterial• Resistance: Ampicillin• Contains lac operon

PRELIMINARY RESEARCH: PLASMID PT-BS

PRELIMINARY RESEARCH: E. COLI K-12

• No plasmids• No toxins (not

pathogenic)• Easy to grow in a

controlled setting (such as a bioreactor for water bioremediation)

• Safe• If it leaks into the

environment, it will die (no chance of contamination)

• Used in the US to produce indigo dye

PCR• Extract

MLHE-1 DNA from lyophilized pellet.

• Conduct PCR to isolate and amplify arxA gene

Restriction Digests• Conduct

restriction digests on the amplicon and plasmid using SpeI.

• PCR purify products

Ligation• Conduct

ligation with purified plasmid and amplicon using T4 ligase.

Transformation• Heat shock E.

coli• Plate

transformed E. coli cells onto LB-Amp-lac plates.

• Pick surviving colonies and subculture

Arsenite Speciation• Test 2 samples

(mutant and control).

• At 0 hr, add 25 ppm sodium arsenite to both samples and take a 5 mL sampling of each to freeze.

• After 48 hours, take a 5 mL sampling of each and freeze.

METHODS OVERVIEW

1. DNA extraction: Extract MLHE-1 genomic DNA from lyophilized pellet using DNeasy Blood and Tissue kit

2. PCR: Conduct PCR (Polymerase Chain Reaction) to isolate and amplify arxA gene. Perform gel electrophoresis to confirm amplification.

3. Restriction Digests and Purify: Conduct restriction digests on the amplicon and plasmid to prepare for ligation using SpeI. PCR purify both products.

4. Ligation: Conduct ligation with purified plasmid and amplicon using T4 ligase. Perform gel electrophoresis to confirm ligation.

5. Transformation: Heat shock E. coli in a calcium chloride solution to make the bacteria incorporate the plasmid with the arxA gene.

MATERIALS & METHODS

6. Subculture: Plate newly-transformed E. coli cells onto LB-Ampicillin-lactose plates. Because plasmid pT-BS contains an ampicillin resistant gene, the colonies that will survive will have the plasmid. Bacteria without the plasmid will die. Pick surviving colonies and subculture.

7. Arsenic Speciation Test: The purpose of the arsenic speciation test is to see if the bacteria effectively converts arsenite to arsenate.• Test 2 samples (mutant and control)• At 0 hr, add 25 ppm sodium arsenite to both

samples and take a 5 mL sampling of each to freeze.

• After 48 hours, take a 5 mL sampling of each and freeze.

• Run samples through arsenic speciation machine

MATERIALS & METHODS

RESULTS: LIGATION GELla

dd

er

ligations

Uncu

t pla

smid

Cut

pla

smid

Overall, the cut plasmid moved faster than the uncut plasmid (the cut plasmid was lighter than the uncut

plasmid), and the ligations moved faster than the uncut plasmid.

RESULTS: TRANSFORMATIONPlates with a

normal amount of Ampicillin, 100

micrograms/mL.

Plates with a lower amount of Ampicillin, 50 micrograms/mL.

All the transformed E. coli grew on LB-ampicillin-lactose plates (6 was control – no growth seen, as expected). Different ampicillin concentrations were used because the scientific paper detailing the plasmid did not specify how much ampicillin the plasmid could resist and still grow on.

RESULTS: ARSENIC SPECIATION

In the E. coli K-12 GMO (with the arxA gene) samples, As(V) concentration increased by 991 µg/L, indicating the production of As(V). An equivalent decrease in As(III) concentration was not seen but the expected decrease (of 991 µg/L) is well within the 10% error margin (i.e. 1910 µg/L in this case) of these observations.

0 Hr 48 Hr0

5,000

10,000

15,000

20,000

25,000

19100 19100

6591650

E Coli K-12 GMO (with arxA)

As(III)

As(V)

µg

/L

RESULTS: ARSENIC SPECIATION

The E. coli K-12 wild type graph indicates that the arsenate concentration decreased by 377 µg/L over a period of 48 hours.

0 Hr 48 Hr0

5,000

10,000

15,000

20,000

25,000

20300 20300

1210 833

E Coli K-12

As(III) As(V)

µg

/L

CONCLUSION

A new bacteria GMO was created that, as indicated by the arsenite test, successfully oxidizes arsenite to arsenate.

After arsenite is oxidized by the bacteria, the arsenate produced can be removed from water with little cost using precipitation.

Although other bacteria have been used as successful bioremediation agents to address heavy metal contamination in water, this is the first study investigating use of the arxA gene for the purpose of arsenic water bioremediation.

APPLICATION: BIOREMEDIATIONCurrent treatments for arsenite removal include filtration, reverse osmosis, or membrane separation, but are often too expensive for practical implementation for small water supplies, and introduce the potential for carcinogenic by-products of the procedures.

Utilizing the mutated E. coli with the arsenite-oxidizing arxA gene as a means to convert arsenite to arsenate (which could then be removed by common methods) would be a low-cost way to reduce the arsenic in polluted groundwater. This could be achieved by building bioreactors in places where drinking water contains high arsenic concentrations, which could improve the quality of life for millions of people.

ACKNOWLEDGMENTS AND REFERENCES

I am grateful to Dr. Aru Hill and Ms. Belinda Schmahl for offering support throughout this project. I am grateful to McCampbell Analytical and Applied Speciation and Consulting for making it possible for me to perform an arsenic speciation test.

• A. Conrad et. al, The role of ArxA in photosynthesis- linked arsenite oxidation by bacteria from extreme environments. Available at http://events.jpdl.com/pdf/e120624aAbstract02719.pdf (15 September 2012).

• J. Stolz, P. Basu, R. Oremland, Microbial Arsenic Metabolism: New Twists on an Old Poison. Microbe.5, 53-59 (2010).

• S. Hoeft, T. Kulp, S. Han, B. Lanoil, R. Oremland, Coupled Arsenotrophy in a Hot Spring Photosynthetic Biofilm at Mono Lake, California. Appl Environ Microbiol.76, 4633-4639 (July 2010).

• R. Oremland, C. Saltikov, F. Wolfe-Simon, J. Stolz, Arsenic in the Evolution of Earth and Extraterrestrial Ecosystems. Geomicrobiology Journal.26, 522-536 (2009).

• T. Kulp et. al., Arsenic (III) Fuels Anoxygenic Photosynthesis in Hot Spring Biofilms from Mono Lake, CA. Science.321, 967-970 (August 2008).

• R. Oremland, J. Stolz, Ecology of Arsenic. Science.300, 939-943 (May 2003).