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Evolution Characterization of Escherichia coli Using RFLP DNA Fingerprinting

by Lee, Chin How; Lee, Kun Cheng; Oon, Jack Si Hao

Abstract (Summary)
Escherichia coli are commonly found in intestine of human and any adaptation or evolution may affect the human body. The relationship between E. coli and food additives is less studied as compared to antibiotics. E. coli within our human gut are consistently interacting with the food additives; thus, it is important to investigate this relationship. In this study, we observed the evolution of E. coli cultured in different concentration of food additives (sodium chloride, benzoic acid and monosodium glutamate), singly or in combination, over 70 passages. Adaptability over time was estimated by generation time and cell density at stationary phase. Polymerase Chain Reaction (PCR) / Restriction Fragments Length Polymorphism (RFLP) using 3 primers and restriction endonucleases each was used to characterize adaptation/evolution at genomic level. The amplification and digestion profiles were tabulated and analyzed by Nei-Li Dissimilarity Index. Our results demonstrated that E. coli in every treatment had adapted over 465 generations. The types of stress were discovered to be different even though different concentrations of same additives were used. Genomic analysis by RFLP shows that the stress response in E. coli is similar. In addition, monosodium glutamate may be a nutrient source and support acid resistance in E. coli.
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Advisor:Maurice HT Ling

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Source Type:Other

Keywords:escherichia coli, adaptation, experimental evolution, food additives

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Date of Publication:02/01/2010

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SINGAPORE POLYTECHNIC

SCHOOL OF CHEMICAL AND LIFE SCIENCES

Diploma in Biotechnology
General Biotechnology Option

Evolution Characterization of Escherichia coli
Using RFLP DNA Fingerprinting

Project Code: DBTBTech0902

Lee Chin How (0716767)
Lee Kun Cheng (0716499)
Jack Oon Si Hao (0716415)

Year of Study: Year 3

Project Supervisor: Maurice Ling

AY2009/2010


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Abstract

Escherichia coli are commonly found in intestine of human and any adaptation or evolution
may affect the human body. The relationship between E. coli and food additives is less
studied as compared to antibiotics. E. coli within our human gut are consistently interacting
with the food additives; thus, it is important to investigate this relationship. In this study,
we observed the evolution of E. coli cultured in different concentration of food additives
(sodium chloride, benzoic acid and monosodium glutamate), singly or in combination, over
70 passages. Adaptability over time was estimated by generation time and cell density at
stationary phase. Polymerase Chain Reaction (PCR) / Restriction Fragments Length
Polymorphism (RFLP) using 3 primers and restriction endonucleases each was used to
characterize adaptation/evolution at genomic level. The amplification and digestion profiles
were tabulated and analyzed by Nei-Li Dissimilarity Index. Our results demonstrated that E.
coli in every treatment had adapted over 465 generations. The types of stress were
discovered to be different even though different concentrations of same additives were used.
Genomic analysis by RFLP shows that the stress response in E. coli is similar. In addition,
monosodium glutamate may be a nutrient source and support acid resistance in E. coli.

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Acknowledgement

First and foremost, we would like to express our heartfelt gratitude to our project supervisor,
Dr. Maurice Ling Han Tong of Singapore Polytechnic for his valuable guidance, advice and
concern throughout the entire project.

We would also like to extend our appreciations to Mdm Sun Wei and Mdm Kanchini
Manivannan, Technical Officers of Microbiology Laboratory; Ms. Ye Song and Ms.
Cheong Yoke Fun, Technical Officers of Life Science Laboratory; from Singapore
Polytechnic for their timely assistance regarding our laboratory work.

Some special thanks were given to the group of friends around us who provided assistance
and showered their encouragement to us: Issac, Xi Ping, Thaddeus, Ezra and their Final
Year Project members.

Lastly, we like to thank Singapore Polytechnic and Singapore Totalisation Board for
financially sponsoring our project, 11-27801-45-2672.

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Table of Contents

Abstract...................................................................................................................................1
Acknowledgement..................................................................................................................2
List of Figures and Tables ......................................................................................................4
Abbreviations .........................................................................................................................5
1. Introduction ....................................................................................................................6
2. Literature Review ...........................................................................................................7

2.1 Sources and Effects of Genetic Variation...............................................................7
2.2 Experimental Advantages of Escherichia coli......................................................10
2.3 Examples of Evolution Experiments with Bacteria..............................................11
2.4 Long-Term Experimental Evolution in Escherichia coli......................................11
2.5 Effects of Chemicals on Bacteria .........................................................................13
2.6 Fitness Definition .................................................................................................14
2.7 Aims and Hypothesis of Project ...........................................................................14
3. Materials and Methods .................................................................................................15

3.1 Extended Viability in Different Media.................................................................15
3.2 Main Culture Experiment .....................................................................................15
3.3 Treatment Swapping Experiment .........................................................................16
3.4 Polymerase Chain Reaction / Restriction Fragments Length Polymorphism ......17
3.5 Data Analysis........................................................................................................18
4. Results ..........................................................................................................................24

4.1 Different Media ....................................................................................................24
4.2 Generation Time...................................................................................................26
4.3 Day 5 and Day 7 Cell Density..............................................................................29
4.4 Swap Experiment..................................................................................................34
4.5 Polymerase Chain Reaction / Restriction Fragment Length Polymorphism........39
5. Discussion.....................................................................................................................41

5.1 Nutrient Broth Sustains Continuous Growth Up to 24 Days ...............................41
5.2 Nutrient Broth Does Not Prime Cells for Growth in Other Treatments...............42
5.3 Cells Adapt to Their Individual Treatments.........................................................42
5.4 Cells Adapt After 25 Passages .............................................................................45
5.5 Cells from Different Treatment Becomes Genetically Similar ............................46
6. Recommendations ........................................................................................................48
7. Conclusion....................................................................................................................49
8. References ....................................................................................................................50
Appendix A – Extended Bacterial Culture in Different Medium.........................................55
Appendix B – Number of Generations.................................................................................56
Appendix C – Generation Time Estimation .........................................................................63
Appendix D – Cell Density at Stationary Phase...................................................................64
Appendix E – Swap Treatments ...........................................................................................74
Appendix F – Gram Staining Pictures..................................................................................76
Appendix G – Agarose Gel Electrophoresis of PCR-RFLP.................................................78
Appendix H – Dissimilarity Matrix....................................................................................156
Appendix I – Bactome: A module for analysing bacterial omics.......................................158

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List of Figures and Tables

Figure 3.1: Complete Experimental Design
Table 3.1: Effects of the 28 pair-wise comparisons among the 8 treatments. The shaded
areas represent the individual effects of the comparisons.
Figure 4.1: OD600 readings of Escherichia coli in Nutrient broth (NB), Luria-Bertani broth
(LB) and Brain Heart Infusion (BHI) cultured over 41 days.
Figure 4.2: Viable plate count of Escherichia coli in Nutrient broth (NB), Luria-Bertani
broth (LB) and Brain Heart Infusion (BHI) cultured over 41 days.
Figure 4.3: Generation times of the eight treatments: High MSG (A), Low MSG (B), High
BA (C), Low BA (D), High Salt (E), Low Salt (F), High COMB (G) and Low
COMB (H) across 70 passages arranged according to the stress level from highest
to lowest based on the gradient of the linear regression line.
Table 4.1: Tabulation of Coefficient of Variation of all treatments for 70 passages.
Figure 4.4: Coefficent of Variation of all treatments for 70 passages.
Figure 4.5: Ratio of Day 7 to Day 5 cell density of High and Low MSG treatments over 70
passages.
Figure 4.6: Ratio of Day 7 to Day 5 cell density of High and Low BA treatments over 70
passages.
Figure 4.7: Ratio of Day 7 to Day 5 cell density of High and Low Salt treatments over 70
passages.
Figure 4.8: Ratio of Day 7 to Day 5 cell density of High and Low Combination treatments
over 70 passages.
Figure 4.9: Generation Time Trend of Low Salt Treated Cells into Non-Salt Treated Media
(High MSG, Low MSG, High BA, Low BA, High Combination and Low
Combination) over 12 Swaps.
Figure 4.10: Generation Time Trend of Low Treatment Cells into High Treatment Media
over 12 Swaps.
Figure 4.11: Generation Time Trend of High Treatment Cells into Low Treatment Media
over 12 Swaps.
Figure 4.12: Generation time of High Treatment Cells to High Combination Media for 12
swaps.
Figure 4.13: Generation time of Low Treatment Cells to Low Combination Media for 12
swaps.
Figure 4.14: Dissimilarity index of the 28 pair-wise comparisons for the 6 PCR/RFLP.
Figure 4.15: Estimation of the average maximum and minimum mean values of the DI for
each PCR/RFLP count.
Table 4.2: Tabulation of P-value for the resulting effects.

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Abbreviations
BSA Bovine Serum Albumin
BHI Brain Heart Infusion
CC Correlation Coefficient
DI Dissimilarity Index
DNA Deoxyribonucleic Acid
dNTPs Deoxyribonucleotide Triphosphates
EDTA Ethylenedinitrilotetraacetic Acid
H BA High Concentration of Benzoic acid
H MSG High Concentration of Monosodium Glutamate Treatment
H SALT High Concentration of Salt Treatment
H COMB Low Concentration of Combination Treatment
LB Luria Bertani broth
L BA Low Concentration of Benzoic acid Treatment
L MSG Low Concentration of Monosodium Glutamate Treatment
L SALT Low Concentration of Salt Treatment
L COMB Low Concentration of Combination Treatment
NB Nutrient Broth
SNP Single Nucleotide Polymorphisms
STE Sodium Chloride–Tris–EDTA

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1. Introduction

The Gram negative bacterium, Escherichia coli, is commonly found in intestine of human.
As a normal flora of the gut, E. coli can benefit the host by producing Vitamin K or
preventing pathogenic bacteria growth in the intestine. However, these bacteria may adapt
or evolve which may affect the human body.
E. coli evolution to antibiotics tolerance and resistance are widely studied but the
mechanisms to non-antibiotic agents, such as preservatives are less understood. Food
additives are commonly used in limiting microbial growth and flavoring in various types of
food products. Since E. coli within our human gut are consistently interacting with the food
additives, it is important to investigate their relationship.
This project aimed to observe the adaptation of E. coli cultured in different concentration of
food additives, namely sodium chloride, benzoic acid and monosodium glutamate. E. coli
cells are cultured in 8 different media over 70 passages and swapped at intervals among the
treatments. Adaptability over time is estimated by generation time and cell density of
stationary phase. Polymerase Chain Reaction (PCR) and Restriction Fragments Length
Polymorphism (RFLP) are also used to characterize adaptation/evolution at genomic level.

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2. Literature Review

Evolution is the result of genetic changes within a population from one generation to the
other. These changes refer to the modification to the DNA sequence resulting in genetic
variation. The genetic traits in the individual are inherited down from one generation to the
other. As these individual genetic changes accumulate over time, a population can be
formed through the process of genetic divergence (Lenski et al., 1991). These traits may
vary within population and show heritable difference of the organisms. Genetic changes
originated in any generation are usually small and the difference accumulated in each
successive generations can caused substantial changes in the population. Eventually, new
species may be emerged from the ancestor (a speciation event).

2.1 Sources and Effects of Genetic Variation

There are three sources of genetic variation: mutation, gene flow and sexual reproduction.
A mutation is defined as a permanent change in the DNA sequence of the gene, ranging
from a single nucleotide base to a large portion of a gene sequence. The accumulations of
many mutations result in evolutionary changes of the population. There are two ways in
which gene mutation can occur. The first way is by inheriting the mutation from a parent;
the process is known as hereditary mutation. This means that the gene mutation is passed
from the parent to the offspring and the next successive generation will contain the genetic
variation. The second way is that mutation can be acquired during the lifespan of the
organisms. Due to environmental, chemical or physical stress, acquired mutation may occur
to improve the survivability and adaptability of the organism (Travisano, 1997).

Gene flow is defined as the transfer of allele of genes from one population to the other
(Morjan and Rieseberg, 2004). Migration of gene may result in new genetic variants being
introduced to the gene pool of the particular population. For example, species of grass grow
on both sides of the road are likely to transport pollen grains from one side to the other.
When the pollen grains from one side are able to fertilize the grass on the other side and
produce viable offspring, the allele will be successfully transported from one population to
the other. The transferring of genes within or across the population has different effects on

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evolution. Within the population, gene flow can introduce or reintroduce genetic variants to
the population, increase the genetic variation of the organisms. If the gene flow is across
the population, it can make distant population genetically similar to one another
(Buczkowski et al., 2004) which help to reduce the chance of speciation.

Sexual reproduction is the production of offspring with the combination of genetic material
from parental gene which introduces new gene combination into the population, resulting in
diversity. Sexual reproduction is important as it can introduce new combination of gene to
every successive generation which increases the ability of a species to evolve (Colegrave
and Collins, 2008). This implies that advantageous traits from the parental gene may be
combined together and transferred to the offspring. However, there is also a possibility that
good combination of genes may be removed.

The two main mechanisms responsible for evolution are natural selection and genetic drift.
The process whereby the heritable traits are passed on over successive generation to
improve the survivability of organism is known as natural selection (Hurst, 2009). For
natural selection to occur, there are two essential requirements to be met. Heritable
variation for the particular traits must be present and able to exist within the population. In
addition, there must be differential survival and reproduction associated with the possession
of that trait. Through natural selection, the advantageous traits are passed on to the next
generation and more offspring will be able to survive and adapt better. On the other hand,
the trait that does not confer an advantage is unlikely to be passed over to the next
generation.

An example (Saccheri et al., 2008) will be peppered moths (Biston betularia) in England.
The original peppered moths were light gray which blended in with the tree trunk.
However, during the industrial revolution, many industrial released huge amount of air
pollutant. With change in environment, the camouflage of the original moth loses its
function because the tree trunks are covered with air pollutant and turned darker. The dark
gray peppered moths which once at disadvantage and eaten by predators, now survived and
bred while their lighter counterparts were eaten up. Through natural selection and adaption

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over a period of time, the peppered moths eventually change from light gray to dark gray to
match the colour of the tree trunk.
The central concept of natural selection is the evolutionary fitness of an organism (Orr,
2009). The fitness refers to the proportion of subsequent generation that contains the genes.
This concept measures the organism survivability and reproducibility, determining the size
of its genetic contributed to the next generation. For example, if an allele of one gene
confers better fitness over the other allele in the population, this allele will be selected and
passed over to the next generation (Lenz et al., 2009). Subsequently, this particular allele
will become more common within the population after each succesive generation.

The second mechanism for evolution is genetic drift (Mank et al., 2009). Genetic drift is the
change in the frequency of a gene variant occurring in a population due to random sampling.
As compared to natural selection which determines the genetic variant due to successive
generation, genetic drift randomly determines the variant for the next generation and is not
affected by physical, chemical or environmental stress. The variant randomly selected may
be beneficial (Mank et al., 2009), neutral (Bloom et al., 2007) or even detrimental
(Munguia-Vega et al., 2007) to the next generation.

Genetic drift can have several important effects on evolution. The drift will stop eventually
when an allele disappeared from the population or replaced the other alleles entirely. This
mean that the genetic variation in the populations is reduced and the population’s ability to
evolve in response to new stress may be lowered. Another issue is that the effect of the
genetic drift is larger in small population and smaller in large population (Otto and
Whitlock, 1997). Genetic drift occurs faster and has more drastic impact in smaller
population. Thus, rare and endangered species which exist in a smaller population will be
affected most by the drift. Genetic drift can also contribute to the process of speciation
(Devaux and Lande, 2008). Through the process of genetic drift, there is possibility that a
small isolated population will be diverged from the larger population.

Evolution affects the behavior of the organisms and influences every aspect of them. The
outcomes of evolution can lead to adaptation, extinction and speciation. Adaptation is the
process whereby an organism change in behavior, physiology and structure to become more

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