Entry into the Stockholm Junior Water Prize 2011

Monitoring Water Quality Through PCR

Jadelle Riski, Briana Tobin

Alaska

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Abstract

Bacteria from sediment basins on east 68th and Meadow street in Anchorage, Alaska were

examined through SDS-PAGE and PCR. Observations of the basins led us to ask, Could pathogenic

bacteria pose a potential risk to our waterways? Bacteria was grown from water samples taken, then

extracted bacterial proteins, than processed through SDS-PAGE and PCR to compose a verifiable

fingerprint of bacteria. Then samples where sent to University of Alaska Fairbanks to be sequenced and

verified.

SDS PAGE didn’t work as a verifiable fingerprint leading us to use a PCR fingerprint. Results

from UAF where returned, E-Coli as our control was verified, one sample was sequenced as

Paenibacillus (soil, water, vegetation, and insect larvae), the final sample could not be sequenced.

Paenibacillus bacteria is affective for the surrounding environment of the basins, it breaks down

sediment to filter more effectively, so that large substances do not lead to our creeks. This result helps us

to verify that there are also affective bacteria in the sediment basins but doesn’t eliminate the possibility

of pathogenic bacteria in our creeks.

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Table of Contents

Overview

Abstract……………………………………………………………………………………………………1

Key Words...................................................................................................................................................3

Abbreviations and Acronyms......................................................................................................................5

Acknowledgements......................................................................................................................................6

Biography.....................................................................................................................................................7

Introduction..................................................................................................................................................8

Methods........................................................................................................................................................9

SDS-PAGE....................................................................................................................................10

DNA Extraction and Purification...................................................................................................10

Bacterial PCR.................................................................................................................................10

Sequencing Results....................................................................................................................................11

Discussion..................................................................................................................................................11

Conclusion and Future Plans.....................................................................................................................12

Annex 1.....................................................................................................................................................13

Annex 2………………………………………………………………………………………………….14

References................................................................................................................................................15

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Key Words

Sediment Basins---The purpose of Sediment Basins are to have sediments settle to the bottom of the

basins so that clean water can flow over as a filtration system from water runoff of streets and

community drains.

Campbell Creek---Campbell Creek is an anadromous fish stream that flows out of the Chugach

Mountains, through the heart of the City of Anchorage and empties into Cook Inlet. Relatively

unique among urban waterways, Campbell Creek still supports natural runs of four species of

salmon: chinook, coho, pink, and sockeye. Macroinvertibrates---Small organisms without a backbone.

Bacteria---Single-celled or noncellular spherical, spiral or rod-shaped organisms lacking chlorophyll

that reproduce by fission.

Pathogens---Disease producing agent.

Waterways---Bodies of moving water.

SDS PAGE---Protein fingerprint through electrophoresis.

DNA---Genetic makeup of living organisms.

PCR---A DNA Fingerprint through electrophoresis.

Biohazard---Material that is Hazardous to biological organisms.

Cytoplasmic Proteins---Proteins that are found inside the cell

Polyacrylamide Gel---A Polyacrylamide Gel is a separation matrix used in electrophoresis of

biomolecules, such as proteins or DNA fragments

Electrophoresis Chamber---A chamber that draws substances through electrophoresis gels with an

electric current.

West and Weir---Two locations around the sediment basins. Water from the West side of the cities

drainage area leads into the ponds, and the Weir is where the water leads out after filtration into

Campbell Creek.

Ribosomal---Ribosomes are the components of cells that make proteins from amino acids. One of the

central tenets of biology is that DNA makes RNA, which then makes protein

Primers---Sequences a specific section of DNA or RNA.

Oligos---A short nucleic acid polymer, typically with twenty or fewer bases.

Agarose Gel---Agarose gel electrophoresis is a method used in biochemistry and molecular biology to

separate DNA or RNA molecules by size. This is achieved by moving negatively charged

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nucleic acid molecules through an agarose matrix with an electric field (electrophoresis).

E-coli---Bacteria naturally occurring in the lower intestine of many animals, including humans.

BioPrep Program---Alaska BioPREP is a program designed to engage 7th through 12th grade students

and teachers, with emphasis on rural students of Alaska Native ethnicity, in biomedical research

projects that lead toward health careers. This program expands our Alaska community of science

educators and learners. It begins with the intellectual rigor of scientific inquiry and then

addresses the attitudes and social values conducive to learning science. Pilot projects have

proved that if we can engage students in rural Alaska high schools, they go on to science majors

in college.

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Abbreviations and Acronyms

SDS PAGE---Sodium dodecyl sulfate polyacrylamide gel electrophoresis

DNA---Deoxyribose Nucleic Acid

PCR---Polymerase Chain Reaction

rDNA---Recombinant DNA (Deoxyribose Nucleic Acid)

NIH BLAST---National Institute of Health Basic Local Alignment Search Tool

AK---Alaska

(number) C---Celsius

LB---Luria-Bertani Broth

Ect---Et cetera

(number) S---Subunit

(number) F---Forward

(number) R---Reverse

(number) BP---Base Pairs

GF---General Flora

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Acknowledgements

We would like to thank our science teacher Aaron Kallas, for his direction, guidance, and

assistance. Aaron Kallas worked extremely hard, getting the resources we needed with the help of the

University of Alaska Fairbanks through their BioPrep program.

We would also like to thank Christina Young for helping us select the sample we were working

with for this project and for many more still to come.

A special thanks to all the people that encouraged us to go out and help the community, inspiring

us to go forward with this project with our whole hearts to help people in our community, raising

awareness about the waters that play such an integral part in our lives today.

Words cannot express how appreciative we are to our families. The support and guidance they

have given us has been a tremendous encouragement. To all we are eternally grateful. Many thanks.

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Biography

Jadelle Riski and Briana Tobin are outgoing juniors at Polaris K-12 School, where they push

themselves beyond the limits by taking independent courses and extracurricular activities. They are

enthusiastic about environmental sciences, and travel around Alaska to study these sciences. They were

born and raised in Alaska, where they love to hike, bike, fish, and much more. When it comes to

environmental awareness issues, their minds are sparked and the inner scientist reveals it self. In the

future they would like to go to college to study for a degree in environmental biology in order to give

back to the community.

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Introduction

The streams of Alaska are important to all living creatures in Alaska. Campbell Creek is a

local stream in the Municipality of Anchorage Alaska, and is one of the streams that support the

Anchorage community and the wildlife within the area. Campbell Creek is home to many diverse

activities: children playing in the streams, families inner tubing down the creek, salmon living,

macroinvertibrates residing. But with the conditions of the creek today, it raises the question whether the

streams are safe for life in our community.

This project developed from a previous project in August 2009 where we monitored heavy

metals in the sediment basins near the intersection of Meadow Drive and 68th Street in Anchorage,

Alaska (Brayton Sediment Basins)[see appendix 1]. The initial project goal was to investigate any

potentially harmful chemicals and metals that may have leached into Campbell Creek, possibly harming

the macroinvertabrates of the streams [see appendix 2]. Results from testing indicated that harmful

chemicals were being put into Campbell Creek. This project raised the awareness of many. While doing

this project, we worked with the Anchorage Waterways Council, Alaska Fish and Game, and University

of Alaska, Anchorage.

The August 2009 project, led us to believe from our results in the chemical analysis we

conducted that more than inorganic substances in the basins, which could pose a potential risk to the

local waterways. These sediment basins lead into Campbell Creek, which houses many of Alaska’s

macroinvertabrates, wild salmon, and other wildlife. The Creek leads straight into our oceans (NOAA

Community-Based Restoration Program, pg. 1). While taking samples, we noticed there was a

significant amount of substances floating on the water that seemed unusual or un-safe for the area: oil,

garbage, orange residue (possibly iron), and other unrecognizable substances.

A year and a half of observation and sample collecting, led to an investigative question: could

pathogenic bacteria pose a potential risk to our waterways? The problem is that bacteria can be difficult

to monitor for two reasons: bacteria types may have many different strains with some being common

and benign while other potentially hazardous or pathogenic, and it is difficult to positively identify any

pathogenic bacteria without complex equipment and protocols or the expense of sending samples to

labs.

The plan for this project is to explore a possible method of identifying pathogenic bacteria in

local waterways quickly, easily, and less expensively. This method could potentially lead to developing

a mechanism to monitor for harmful pathogens in the basin area and possibly in other basins in

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Anchorage as well. Ultimately, this information could lead to active monitoring of water sources to

ensure that the streams of Anchorage and to other areas in Alaska are healthy. In this way all plants,

animals, and humans can have safe waters.

There will be two main components to this study. The first step is to do a Protein Finger Print, in

which we would collect water samples from the sediment basin and grow a bacteria library from the

water samples. Purify the strains on the LB plates that grow and extract cytoplasmic proteins from the

samples with Laemmli Buffer (Laemmli, 1970) and use SDS PAGE (sodium dodecyl sulfate

polyacrylamide gel electrophoresis) to produce a fingerprint, to isolate the proteins and to determine a

unique fingerprint for each of the bacteria samples (Campbell, 1996).The SDS-PAGE is affordable and

easier to do a procedure, verses identifying solely on genetic information.

The second step is to positively identify the bacteria. We will extract and purify the bacterial

DNA, use specific primers to isolate the universal DNA region 16s rDNA (Chakrabarti, 2009) and use

PCR, a group of DNA to be sequenced, to amplify the region (Mullis, 1986). Finally the samples will be

sent to the University of Alaska Fairbanks for sequencing. The sequenced results will then be compared

against the NIH BLAST (Basic Local Alignment Search Tool). The results will positively identify our

bacterial strains.

By completing this project, our hope is to verify a method of monitoring bacteria based on SDS-

PAGE protein fingerprints rather than the more costly methods of bacterial identification of PCR. PCR

is not only more expensive, by almost twice as much as SDS-PAGE, but also very time consuming

because of the sequencing process of sending it to the university and then having to wait for the

sequence. In the end it takes several weeks to get the samples finished, compared to the minute time of

the SDS-PAGE.

Methods

Samples where taken from the sediment basin at 68th and Meadow, Anchorage, AK located off

of Brayton drive, in early fall of 2010. These samples were frozen for processing in the beginning of

2011.

In January 2011 the samples were randomly selected by a student unrelated to the project, out

of a bag of all the samples collected, for the reason of possibly being bias of picking samples. The plates

were streaked and incubated at 37C for three days to allow the bacteria to grow and create a bacterial

library of each sample. The bacterial types that were grown were then re-plated on separate LB plates in

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Image 1: 1‐Standards, 2‐Ecoli HB101, 3‐General Flora, 4‐West, 5‐Weir, 6‐Additional Standard 

order to purify strains. During the entire process we used safety equipment such as rubber gloves,

goggles, aprons, etc. along with bleach and biohazard bags.

SDS-PAGE

Using Laemmli SDS-PAGE method cytoplasmic proteins where extracted from bacterial

strains and samples were loaded into a 4-15% polyacrylamide gel in a vertical electrophoresis chamber.

Gels were ran and subsequently stained using a coomassie stain then destained using a universal destain.

It was quickly realized that the product did not work; proteins could not be seen in many of the extracted

samples (Image 1). Our idea for a visual reference to the SDS page was not going to work for our

references. Since the discovery of this problem, the decision was to keep to the original plan of verifying

strains through PCR, even though PCR is not the most cost effective way of identification it will be our

default protocol. Still the idea of being able to monitor basins using DNA is possible - but this option

will be more time consuming and costly then the SDS PAGE.

DNA Extraction and Purification

The next process was to verify DNA from the bacteria. This was done by extracting and

purifying the DNA through Qiagen DNEasy Blood and Tissue kit (50) Cat. No. 69504.) The extractions

were successful except for the Weir sample, the sample was cloudy compared to the other sample that

we purified.

Bacterial PCR

From the review of literature, it was found that there existed a well known region of ribosomal

bacterial DNA used for universal identification, 16s rDNA, by using the reliable set of primers were 27F

and 1525R, and ordered oligos from Eurofins Operons.

Formulation was as follows (Table 1):

1       2        3        4          5           6 

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Table 1: PCR Procedure

Sequencing Results

The expected fragment size on a successful PCR reaction to be approximately ~1500bp.

Test gel showed E-coli, GF and West all successful at ~1500 bp. But the Weir sample did not show

successful PCR. Concluding that this failure was due to the possibility of an unsuccessful DNA

extraction that was preformed earlier in the process.

PCR product was sent to the University of Alaska Fairbanks through the BioPrep program to

be sequenced. The results were returned from University of Alaska Fairbanks and subsequently ran

through NIH BLAST (Basic Logic Alignment Search Tool). The table below shows the information

received from the results (Table 2):

Table 2: UAF Sequenced Results

Discussion

From the analysis of our results we found that our water sample contained an affective type of

bacteria for the sediment basins. From our research, Paenibacillus is soil, water, vegetative matter, insect

larvae, all these help to break down the sediment in the basins to which then achieves better filtration.

This shows there are beneficial bacteria in the basins, not just harmful substances in our waters. The

result of these tests still does not eliminate the possibility of other potentially dangerous bacteria. These

potentially pathogenic bacteria could cause a risk to our waterways. Since only one sample out of the

two bacteria water samples could be analyzed, we cannot verify at this point if there is either just

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beneficial or just harmful bacteria or both in the sediment basins. Through further research we can

potentially answer our question “Could bacteria pose a potential risk to our waterways?” and creating a

catalog for others in the community to use.

The problems faced in this project were that the SDS PAGE did not succeed and would not be

able to be referenced back for the future catalog. With this we failed to support our hypothesis by not

being able to identify the bacteria quickly, easily, and affordably through SDS-PAGE. Instead the plan

had to be changed and ran through the procedure of PCR. The variables to be considered for the results

of SDS PAGE are; the samples were taken in August of 2010 and then frozen, then further processed in

January of 2011. Due to the freezing of the samples, we believe there is a chance that the potential of

more bacteria in the water was ruined during the freeze time or was affected by the freeze time and

reacted differently by the substances used with the tests.

Conclusion and Future Plans

Once we create a catalogue of different bacteria we have found using PCR, we found was the

most effective to make a reference for furthering our project. After the conclusion of our findings in this

phase of our project, we plan on working in collaboration, with an environmental middle school science

class to monitor the ponds. These students would go out to the basins and take water samples while

learning about the local environment and ecology of Campbell Creek as well as the surrounding

Sediment Basins. They would at that time be able to grow bacteria colonies and bring the agar bacteria

plates to us so we would be able to sequence them referring to our catalog of bacteria from the sediment

basins we previously created. By this method we can tell the students/and their teachers what bacteria

they had found in the basins. Hopefully this way we will be able to identify and possibly find more types

of bacteria. By doing this we would be bridging gaps in our community, sparking the minds of young

scientists learning about the environment, and monitoring the sediment basins all at once. Our goal is to

eventually benefit, with this data, Alaska Fish and Game management and other environmental groups

in our area. We hope to educate others and bring awareness about bacteria in our water leading to

Campbell Creek and in the end preserve our waterways. The purpose of this study is to protect and

restore the waterways of Anchorage, AK and hopefully our ocean environments of Alaska as a whole

and to educate and inspire our future scientists.

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Annex 1: Sediment Basin Location

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Annex 2: Flow of Campbell Creek

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References

Books

Campbell N, Biology, 4th edition, Benjamin/Cummings Publishing Company, Inc., Menlo Park

(1996)

Chakrabarti P. et al., Application of 16s rDNA based seminested PCR for diagnoses of acute

bacterial meningitis, Indian J Med Res 129, February 2009, pp 182-188.

Laemmli UK, Cleavage of Structural Proteins During the Assembly of the Head of

Bacteriophage T4, Nature 227, 680-685 (1970)

Lu JJ, Perng CL, Lee SY, Wan CC. Use of PCR with universal primers and restriction

endonuclease digestions for detection and identification of common bacterial pathogens in

cerebralspinal fluid. J Clin Microbiol 2000; 38: 2076-80

Mullis KB et al., Enzymatic amplification of DNA in vitro: the polymerase chain reaction, Cold

Spring Harb Symp Quant Biol 51, 263-273 (1986)

Saiki RK et al., Primer-directed enzymatic amplification of DNA with a thermostable DNA

polymerase, Science 239, 487-491 (1998)

Web Pages:

http://campbelltrail.muni.org/CCIT/07-Page.html

Campbell Creek Interpretive Trail

http://www.fakr.noaa.gov/habitat/restoration/CampbellCreek.pdf

NOAA Community--Based Restoration Program---Campbell Creek Restoration

http://wms.geonorth.com/public_education/PublicEducation.aspx#LittleCampbellCreek

Anchorage Watersheds and Water Quality Science