The Use of Molecular Techniques to Differentiate Escherichia coli Strains and Identify Genomic Changes in Evolving Populations of E. coli cells




Catherine Ruggiero

Introduction

Evolution can be monitored by examining changes in the genomes of populations. Detecting these changes requires a molecular technique that allows us to highlight differences between samples of genomic DNA. Ideally, such methods would also enable us to identify areas of the genome where changes have occurred in order to elucidate the type of change that has taken place (i.e. inversions, deletions, changes in transposable element copy number, etc.).

RAPD is a PCR-based analysis that uses small arbitrary primers to produce random PCR products. RAPD analysis has been used in E. coli to differentiate isolates from different sources, identify different strains, and even detect genetic rearrangements due to environmental stressors. Therefore, it may be a useful tool to investigate evolutionary change between populations.

This experiment utilized the RAPD methodology to investigate differences among evolving populations of E. coli. In order to investigate whether genomic changes could be detected in evolving populations of E. coli, I first showed that RAPDs can be used to differentiate between closely related strains of E. coli (JM109 and K12). Once this was established, I used RAPD analysis to do a preliminary investigation into the differences between populations of K12 E. coli that have been evolving in the lab. By examining populations that had evolved for at least 2,000 generations under different conditions, as well as the starting population they evolved from, I was able to test whether the RAPD methodology could distinguish between samples of the same strain at different evolutionary time points.

Figures


Figure 1-RAPD analysis of JM109 and K12 E. coli strains using primers OPA-01 through OPA-10 (primer used is indicated at the bottom of gel pictures). Differences between the strains were detected using primers OPA-04 and OPA-07. All other primers showed identical banding patterns between the two strains.


Figure 2-RAPD analysis of JM109 and K12, Generation 0 E. coli DNA were run with K12 small and large evolved population samples. Evolved populations all originated from K12, Gen. 0 and had been evolving under conditions that kept the population size small or large. Primers OPA-04 and OPA-07 were used. Differences between the two strains matched previous findings. There were no differences detected between the two evolved populations (small and large) or between the evolved populations and their starting population (K12, Gen. 0). Numbered bands were sequenced. Band 4-2 was the same fragment in all samples (JM109 and K12), as were bands 7-2 and 7-3 (same fragment). Band 7-1 (in JM109 only) matched F-plasmid DNA, which has been removed from the K12 strain. See figure below for information on bands 4-1 and 7-4.


Figure 3-Sequence information for bands 4-1 (A) and 7-4 (B) showed where the amplicon was located in the E. coli genome. These bands were present in the K12 strain only and were absent from JM109. Possible explanations for this include: no sequence homology between JM109 and the primers at these locations, a rearrangment at one or both primer positions in JM109, or an insertion between the primers in JM109.


The use of molecular techniques is critical to detecting evolutionary change in populations. Random Amplified Polymorphic DNA (RAPD) analysis was performed to see whether genomic differences could be identified in evolving populations of E. coli. The methodology was first applied to two different strains of E. coli (JM109 and K12) to confirm that the RAPD analysis was capable of detecting differences in closely related genomes. Following confirmation that this technique possessed the strength to differentiate the strains, RAPD analysis was performed on DNA extracted from evolving populations of E. coli cells. Sequencing information identified the DNA amplified by RAPDs as homologous to E. coli and also illustrated where differences between the two strains were located in the genome. Although genomic differences among evolving populations of a single E. coli strain were not identified here, I conclude that RAPD analysis can be used to differentiate between very similar genomes and could be useful in detecting evolutionary changes among populations of the same species or strain.

Full Paper

Acknowledgments

I would like to thank Dr. Berish Rubin for his advice and guidance throughout this project. I would also like to thank Bo Liu and Leleesha Samaraweera for their constant assistance and insight.


This document was last modified 05/12/2009.
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