Biology

The Power of PCR

Abstract

The purpose of this report is to demonstrate the link, or lack thereof, between the size of the yecG-flhDC intergenic region and motility in Escherichia coli. Motility in E. coli is important as it dictates whether infection with the bacterium can spread within the body.

This was completed through the screening of a biobank of 183 strains of E. coli, which were collected through a clinical study, via Polymerase Chain Reaction (PCR). This screening was accomplished via growing the samples on LB plates, inoculating LB cultures, isolating the DNA through a culture-to-test-tube preparation, and then setting up and running a PCR. The products were analysed through gel electrophoresis and the size of the yecG-flhDC region observed.

 

 

Introduction

UTIs(Urinary Tract Infections) are bacterial infections of the urinary tract that are caused by E. coli in about 80-90% of cases (Behzadi, et al. 2010). The term encompasses infections of the bladder and lower urinary tract (cystitis), in addition to infections of the kidneys and ureter. The main symptoms are a frequent urge to urinate and burning pain when urinating. Upper urinary tract infections can also cause pain in the sides and back, nausea, and a high temperature. Age, female sex and diabetes are risk factors (National Health Service 2016) .

UTIs are very common; 3% of all GP visits in the UK are UTI related, and the majority of women will contract a UTI at some stage in their life (General Practice Notebook 2016). UTIs are treated with a 3-day course of antibiotics, the cost of treatment for one UTI to the NHS is approximately £33.40. This translates to about 10,200,000 GP visits related to UTIs annually in the UK, and about £340 million in expenditure (British Medical Association 2014). This is why UTIs are of scientific interest.

Current perception is that the size of the yecG-flhDC intergenic region controls motility in E. coli; that if the region is longer, due to the presence of insertion elements as a result of transposon activity (transposons are the sections of DNA which can copy themselves and insert these copies elsewhere in the genome – insertion elements are the copies of these sections), then E. coli will be motile. Previous articles discussing the yecG-flhDC intergenic region (the stretch of DNA between two genes in E. coli, called respectively the yecG gene and the flhDC gene) also indicate that it is a hotspot for insertions (Barker, Prüß and Matsumura 2004) (Gauger, et al. 2007). This indicates that this region has a lot more than average transposon activity.

Motility is self-directed movement of the bacterium – it is largely, but not always, due to the presence or absence of a tail-like organelle called a flagellum. Motility is important because motile strains of E. coli are more likely to develop from cystitis into an infection of the upper urinary tract. This type of infection is far more serious, as well as more costly and time-consuming to treat.

Approximately 32% of the samples screened are motile. If the hypothesis that the yecG-flhDC intergenic region controls motility is correct in the context of the strains screened from the ecological environment of a human bladder, then occurrence of the transposon should be approximately 32%.

Methodology

For the PCR, I began by streaking out the 183 strains of E. coli onto LB-agar plates (pictured right); LB stands for Luria Broth, and is a nutrient-rich broth that forms a gel with agar. These plates were then left to grow overnight at 37º Celsius. The following day, LB medium was inoculated with the strains. This was done by aseptically pipetting 3ml of LB into tubes and using an applicator stick to dab a single colony from the LB plates and swirl it in the medium. The cultures (pictured right, below) were then returned to 37ºC on a platform shaker at 150 rotations-per-minute overnight (allowing DNA density to reach an appropriate level) to grow. The following day, they were used in a PCR reaction.

The strains of E. coli used were collected from 30 patients with recurrent UTIs over the course of a 12-week clinical trial. It was important to screen all of these strains to establish the frequency of occurrence of the insertion elements resulting in a longer yecG-flhDC intergenic region.

For all reactions, setting up the PCR involved combining the following to form a PCR mix:

  1. PCR Primers. These are short sequences of nucleotides that provide a starting point for DNA synthesis as DNA polymerase can only act on existing strands of nucleotides. Two primers are used as each acts on one strand of DNA (Nature Education n.d.).
  2. Polymerase. Two different types of polymerase were used over the course of the reaction: Taq and Q5.
  3. Buffer. This regulates pH to provide optimum conditions for polymerase activity.
  4. Deoxyribonucleotide triphosphates: dNTPs. These are single units of bases that are used as ‘building blocks’ for DNA.
  5. PCR water.

Table 1, Volumes for PCR mix

X Volume per reaction (in μl)
PCR Water 27.5
Buffer 10
dNTPs 5
Primer (1430) 2.5
Primer (1431) 2.5
Q5 DNA Polymerase 0.5

The DNA was isolated either by using a kit or a culture-to-test-tube preparation. For the kits, the manufacturer’s protocol was followed.

When a culture-to-test-tube preparation was used, 20μl of the cultures were diluted in 180μl of PCR water and centrifuged for five minutes. This caused the DNA to form a pellet at the bottom of the tubes. The PCR water was removed with a vacuum aspirator (this uses suction to draw up the water into a conical flask for collection and disposal) and 100μl of fresh PCR water added. The tubes were Vortexed (mixed very quickly using an electric motor which causes a central platform to oscillate) to resuspend the pellets. The tubes (pictured right, above) were placed in a heat block set to 100ºC for 10 minutes, and then transferred to an ice bucket. These tubes were then thermocycled (brought up to 90°C, down to 50°C, and back up to 70°C) for approximately two hours.

Whilst the reactions were in the PCR machine, a 0.8% agarose gel was poured with 2μl of Nancy-520. This acts as a stain and is a safer alternative to carcinogenic Ethidium Bromide. Adding Nancy-520 only when pouring the gel prevents the stain degrading from being stored at a high temperature.

The gel (pictured right) was then loaded and run on a horizontal gel electrophoresis unit for 50 minutes at 100V. Gel electrophoresis works through passing a current across the gel so that each end is charged. DNA is negatively charged, so will move towards the positively charged electrode. Smaller fragments of DNA will move through the gel more quickly than larger ones, resulting in bands at different heights depending on the size of particular fragments (Khan Academy n.d.).In one of the wells, 5μl of a DNA ladder was loaded; this is a reference containing DNA fragments with known lengths to compare the samples to (Oppenheimer 2013).

The gel was then observed under a UV transilluminator (pictured right), which uses a fluorescent light to visualise the DNA (UV Transilluminator n.d.).

Results

Previous articles discussing the yecG-flhDC region in E. coli indicate that there was liable to be extensive transposon activity; variation in motility (and in the context of E. coli, development of the largely-harmless cystitis into kidney infections, which are painful, difficult to treat and can have serious consequences such as sepsis) in strains of E. coli is currently attributed to transposon activity in this region.

In the strains screened, this was empathically not the case.

Of these strains, as well as another 300 strains from different ecological environments screened by the lab, only two strains possess the transposon. This suggests an approximately 0.004% occurrence of insertion in the region; no higher than the natural rate of transposon activity. Of the 10,200,000 GP visits related to UTIs annually, only about 40,800 of those UTIs would possess the transposon.

Discussion and Conclusion

In the one strain of E. coli used in the existent literature on this subject (K-12), an insertion in the yecG-flhDC intergenic region results in increased motility, which was shown via motility assays. This involved staining the bacteria, placing them on a viscous agar plate and monitoring their movement over a period of time. In the samples screened, as well as others screened by the lab, there was significant variation in motility. 51% of the samples in the lab’s collection of E. coli strains were motile to some degree. Of the samples analysed that were collected from patients, 32% were motile. Incidentally, the variation in motility from samples from different ecological environments suggests that environment does affect motility in E. coli.

However, very few of these samples showed transposon activity. This demonstrates quite decisively that, within the context of the samples analysed by myself and the lab, variation in motility is not due to transposon activity in the yecG-flhDC region. This raises the question: if not that, then what? There must be other factors influencing motility in E. coli, and the next step would be to establish potential alternative explanations. Only once the cause of motility is established can there be significant progress to establish how to detect and control the spread of motile strains.

 

 

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Acknowledgements

Thank you to Claire Willis, Nuffield Research Placement Coordinator for the North-East, for working so tirelessly to provide opportunity. This one has been appreciated and valued beyond measure. Thank you to Dr Phillip Aldridge, my project supervisor, for agreeing to host us and for being a constant source of support, advice, and coffee throughout my project. Thank you to everyone else at the Centre for Bacterial Cell Biology, the Institute for Cell and Molecular Biosciences, Newcastle University, and the Nuffield Foundation.

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