Biochemistry experiment 4A and 4B – Time course of an enzyme catalysed reaction/ Effect of pH on the rate of an enzyme catalysed reaction

  • Introduction

Enzymes are organic molecules comprising of proteins. They are highly specific in the way in which each one catalyses a unique individual reaction. As enzymes are comprised of proteins they can denature if not kept in idyllic conditions, therefore they lose their function thus stopping any reactions (Gomes and Rocha-Santos, 2018). As enzymes are highly specific they each work differently in varying degrees of pH. (Krogdahl et al., 2015) Enzyme catalysed reaction experiments can be extremely important and beneficial, especially in some fields of medical research. Tapper et al., (2017) suggests that elevated liver enzymes within a patient can indicate detection of a wide variety of liver diseases. With the use of enzyme catalysed reaction experiments, several different tests to determine which type of disease is present can be done all at once. This is a more efficient and affordable way to obtain fast results.

 

  • Materials and methods

 

2.1 Part A: Time course of an enzyme catalysed reaction

5 sets of 3 microcentrifuge tubes were firstly labelled A1-E3. The following solutions were then added to each microcentrifuge tube; 0.2 mL distilled water, 0.25 mL sodium acetate buffer solution (pH5.0) and 0.25mL para-nitrophenol phosphate (pNPP) solution. Each tube was then left to equilibrate at room temperature for 5 minutes, then immediately transferred to a water bath of 30  where they were left for a further 5 minutes. 0.05 ml of enzyme was then added to each tube and gently agitated. In order to maintain accurate timings, the enzyme was added to each tube at 1-minute intervals. The times were recorded by a clock timer. The 5 sets of microcentrifuge tubes were incubated for 5, 10, 15, 20 and 30 minutes (i.e. tube sets A1-A3 incubated for 5 minutes, tube sets B1-B3 incubated for 10 minutes etc). At the end of each incubation period the reaction was stopped by adding 0.5 ml sodium carbonate and the tube was then placed back into the test tube rack. Before determining the absorbance of each reaction mixture, a blank solution was prepared by adding the following solutions into a microcentrifuge tube; 0.2 mL water, 0.25 mL sodium acetate buffer, 0.25 mL pNPP, 0.5 mL sodium carbonate and lastly 0.05 mL of enzyme. To determine the rate of absorption the spectrophotometer had to firstly be set to calibrated with the blank solution. The absorbance of each reaction mixture was measured at a wavelength of 410nm. Before the absorbance could be measured by the spectrophotometer, the contents of each microcentrifuge tube were transferred into a low-volume cuvette.

2.2 Part B: Effect of pH on the rate of an enzyme catalysed reaction

The same basic procedures were followed as mentioned before in section 2.1. Although, for this experiment 4 sets of 3 microcentrifuge tubes were used, with each set containing a different pH buffer. There were 4 different pH buffers provided which included; pH 3, pH 5, pH 7 and pH 9. Each of the 4 sets of microcentrifuge tubes were all placed in a water bath at 30  for 10 minutes. Following completion of incubation, the reaction was then stopped by adding 0.5 mL sodium carbonate to each tube.  The incubation period was chosen as 10 minutes sat in the centre of the linear trend line and this was deemed the most accurate choice. The blank used to calibrate the spectrophotometer in part B was prepared in the exact same way as the blank that was used in part A. After calibration of the spectrophotometer, the absorbance of each reaction mixture was again measured at a wavelength of 410nm. The raw data obtained from the spectrophotometer readings was then converted into the amount of pNP produced within 10 minutes. This data was obtained by using equation 1. Equation 2 was then used to convert the data obtained from equation 1 into the rate of pNP produced per minute. A mean average was then taken from this final set of data to be used to plot the graph that is shown in figure 3.

Equation 1 – pNP produced (μmol)= (absorbance @ 410nm-0.0035)/3.1576

 

Equation 2 – rate of reaction (μmol / min)=(amount of pNP (µmol))/(10 (min))

 

  • Results

To begin the results, the pNP calibration data provided in experiment 4A was converted into a graph as shown by figure 1. The equation provided by this first graph was then used to convert the absorbance values that were obtained in experiment 4A into concentration of pNP produced (µmol).

Figure 1

 

Figure 1 – pNP calibration data plotted to obtain equation y = 3.1576x + 0.0035.

This equation was then used to convert the recorded 3 absorption values for each set of 5 into µmol of pNP produced for each different time frame (5 minutes, 10 minutes etc.). A mean average was then taken from the 3 absorbances samples from each set. This mean average was then plotted on the graph shown in figure 2. The graph was used to calculate an accurate incubation time for the samples used in experiment 4B.

Figure 2

 

Figure 2 – Mean averages taken from the three samples recorded for the 5 different sets, plotted against the time each set was incubated for. The standard deviation from all the results recorded for each of the 5 sets was calculated to be used in the form of error bars to indicate accuracy.

It was decided that 10 minutes would be the optimum incubation time for the samples produced in experiment 4B. This decision was made as 10 minutes sits in-between the linear phase. This was deemed appropriate as 5 or 15 minutes may not have been as accurate as the sat at the very beginning and the very end of the linear phase.

Figure 3

Figure 3 –  The rate in which pNP (µmol) is being produced per minute at 4 different levels of pH, including; pH3, pH5, pH7 and pH9. The samples were incubated for an optimum time of 10 minutes in a water bath at 30.

 

  • Discussion

Acid phosphatase hydrolyses phosphate from a variety of molecules which contain a phosphate group (Wang and Liu, 2018). The substrate used within this experiment was para-nitrophenol phosphate (pNPP). When subjected to acid phosphatase, pNPP is hydrolysed to create para-nitrophenol (pNP). Within an alkaline solution pNP can be detected as it turns yellow in colouration (Jez et al., 2016). This is beneficial as the colouration can be assessed with the use of a spectrophotometer to monitor the rate of production of pNP. Production rate of any enzymes relies on specific environments.

Observing figure 3 it is clear to see that the idyllic pH for acid phosphatase to produce pNP is pH 5. As enzymes are acidic in nature it could be assumed that pH 5 would be optimum as acid phosphatase is clearly acidic in nature so would work best in an acidic environment. pH 5 is deemed acidic without it being too harsh that it becomes destructive to the enzyme. It could also be argued that pH 7 would be ineffective as the solution would still be too neutral for a productive enzyme reaction rate. This argument can be supported by Behera et al., (2017) who also carried out an enzyme assay on acid phosphatase. It is stated within this experiment that the optimum acid phosphatase activity was recorded at a temperature of 48 at a pH of 5. However, although the optimum pH for both experiments was deemed pH 5, two different temperatures were used which would have affected both results. Nevertheless, both arguments can be debated as Jennifer et al.,(2015) suggests that the optimum environment for the production of enzymes is pH 9 at a temperature of 40. The comparison of these arguments could propose that further study should be completed to investigate the effects of varying temperatures on the production of enzymes.

Optimal conditions are essential for enzyme production. A change in environment can cause an enzyme to denature, thus meaning they stop functioning, which will result in a stop of production. (Saoudi et al., 2017) Denaturation is the process in which an enzyme will undergo threatening and even destroying structural change. Although enzymes are subject to structural damage, no reaction would be strong enough to break the peptide bonds between amino acids, so the enzymes primary structure would remain unchanged (Cheng et al., 2015). As the process of denaturation causes the enzyme to change structure, this then means that the specific substrate matched with that enzyme can no longer bind as the active sight region has been impaired resulting in the substrate and enzyme no longer being a match (Witkowska et al., 2018).

 

  • Conclusion

To conclude, the production rate of pNP was measured over numerous lengths of time to investigate the most productive time span for pNP production. This information was then used to evaluate the best incubation time for the solutions used with varying degrees of pH. The experiment deemed that pH 5 was the optimum environment for pNP production. Literature suggests that this is due to the enzyme working well within an acidic environment because it itself is acidic in structure, although the pH concentration wasn’t too acidic that it would cause any structural damage to the enzyme, causing it to denature.

 

 

 

 

References

Behera, B. C., Yadav, H., Singh, S. K., Mishra, R. R., Sethi, B. K., Dutta, S. K. and Thatoi, H. N. (2017) ‘Phosphate solubilization and acid phosphatase activity of Serratia sp. isolated from mangrove soil of Mahanadi river delta, Odisha, India.’ Journal of Genetic Engineering and Biotechnology, 15(1) pp. 169–178.

Cheng, B., Wu, S., Liu, S., Rodriguez-Aliaga, P., Yu, J. and Cui, S. (2015) ‘Protein denaturation at a single-molecule level: the effect of nonpolar environments and its implications on the unfolding mechanism by proteases.’ Nanoscale, 7(7) pp. 2970–2977.

Gomes, A. R. and Rocha-Santos, T. A. P. (2018) ‘Enzyme Assays☆.’ In Reference Module in Chemistry, Molecular Sciences and Chemical Engineering. Elsevier.

Jennifer, E. B. M., Sathishkumar, R. and Ananthan, G. (2015) ‘Screening of extracellular hydrolytic enzymes production by ascidians (Polyclinum glabrum, Microcosmus exasperates and Phallusia arabica) associated bacteria from Tuticorin, Southeast coast of India.’

Jez, J. M., Ravilious, G. E. and Herrmann, J. (2016) ‘Structural biology and regulation of the plant sulfation pathway.’ Chemico-Biological Interactions. (Special Issue on Sulfation Pathways), 259, November, pp. 31–38.

Krogdahl, Å., Sundby, A. and Holm, H. (2015) ‘Characteristics of digestive processes in Atlantic salmon (Salmo salar). Enzyme pH optima, chyme pH, and enzyme activities.’ Aquaculture. (Proceedings of the 16th International Symposium on Fish Nutrition and Feeding), 449, December, pp. 27–36.

Saoudi, O., Ghaouar, N., Ben Salah, S. and Othman, T. (2017) ‘Denaturation process of laccase in various media by refractive index measurements.’ Biochemistry and Biophysics Reports, 11, September, pp. 19–26.

Tapper, E. B., Saini, S. D. and Sengupta, N. (2017) ‘Extensive testing or focused testing of patients with elevated liver enzymes.’ Journal of Hepatology, 66(2) pp. 313–319.

Wang, L. and Liu, D. (2018) ‘Functions and regulation of phosphate starvation-induced secreted acid phosphatases in higher plants.’ Plant Science, 271, June, pp. 108–116.

Witkowska, D., Cox, H. L., Hall, T. C., Wildsmith, G. C., Machin, D. C. and Webb, M. E. (2018) ‘Analysis of substrate binding in individual active sites of bifunctional human ATIC.’ Biochimica et Biophysica Acta (BBA) – Proteins and Proteomics, 1866(2) pp. 254–263.

 

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Osmosis

Water is essential for the survival of all living beings. But, the input and output of water entering the body’s cells must be regulated. This regulation of water is known as Osmosis. Osmosis consists of a solvent and a solution, for example pure water and salt water, being separated by a semi- permeable membrane. This semi – permeable membrane allows water molecules to flow through freely from any direction, whereas the salt molecules will not be able to pass through the membrane as they are too large. Water will always move from the side with the highest water concentration to the side with the lowest water concentration, this is to equalise the concentration of constituents on both sides of the membrane. (Borg, 2017). Osmosis is known as a special type of diffusion. Osmosis is needed within animals as it keeps the water input and output within the body regulated, but osmosis itself also must be regulated. This is known as osmoregulation. Osmoregulation is responsible for monitoring the physiological processes to help maintain a concentrated balance of the body fluids inside and outside the cell. (Osmoregulation – an overview | ScienceDirect Topics, n.d.)

Water can enter the body in a variety of different ways for example; drinking, eating and living in an aquatic environment. As water can enter the body, it can also be lost from the body through; urine, faeces, vomit, lactation etc. Species have evolved over time to keep their cells alive through osmosis and osmoregulation. For example, marine fish are hypotonic to their environment. This means that their body fluids contain more water molecules and less solute molecules in comparison to their environment, which is sea water. (BioBook | Leaf: Why is osmosis so important in biology?, n.d.) If marine fish had not evolved and adapted to their environment, their cells would burst due to swelling after taking in too much water. Baldisserotto et al.,( 2007) states that as marine fish are hypotonic to their environment and lose a lot of water, they counteract this by engulfing sea water. 60-85% of this seawater is then absorbed by the intestine. The oesophagus of marine fish absorbs a lot of the salt, but it is almost impermeable to water. This makes the salt level of the water in the intestine much lower, resulting in more water and less salt being absorbed by the intestine and excess salt being excreted in any small amounts of urine that is produced. This is an adaptation that has been developed by marine fish to respond to water lose due to their environment.

On the other side of this example, there are also species that are hypertonic to their environment, such as fresh water fish. This means that the cells have a higher salt concentrations than the surrounding environment. (BioBook | Leaf: Why is osmosis so important in biology?, n.d.) Fresh water fish are opposite to marine fish so they take in water as their cells are constantly losing water. If the fish did not take in this extra water their cells would shrink up and die. (Garrey, 1916).

Terrestrial animals have numerous ways of conserving water though osmosis, such as waterproof skin, feathers fur etc. One of the more advanced adaptations for conserving water is the kidneys. The Loop of Henle resides within each nephron within the kidneys. It consists of a descending limb and an ascending limb. The descending limb is impermeable to solvents, but water can move around freely, this is where the water is absorbed back into the cells. Due to the absorption of water in the descending limb previously, it results in the filtrate in the ascending limb to become concentrated. Within the ascending limb, some solvents are reabsorbed but it is moderately impermeable to water. Any extra waste is then transported to the bladder and excreted as concentrated urine. The more dehydrated the animal is the more concentred the urine becomes. (Regulation of Water Balance, n.d.)

In summary, the evidence shown within this report reflects osmosis as a necessity for life within a whole organism. Species and cells have developed over time so that their bodies can remain hydrated in the most efficient way possible. Osmosis can be displayed differently throughout a variety of species, all with their own similar yet vastly different adaptations.

 

References

Baldisserotto, B., Mancera Romero, J. M. and Kapoor, B. G. (eds) (2007) Fish osmoregulation. Enfield, N.H: Science Publishers.

BioBook | Leaf: Why is osmosis so important in biology? (n.d.). [Online] [Accessed on 3rd December 2017] https://adapaproject.org/bbk_temp/tiki-index.php?page=Leaf%3A+Why+is+osmosis+so+important+in+biology%3F.

Borg, F. (2017) ‘What is Osmosis? Explanation and Understanding of a Physical Phenomenon.’

Garrey, W. E. (1916) ‘The Resistance of Fresh Water Fish to Changes of Osmotic and Chemical Conditions.’ American Journal of Physiology — Legacy Content, 39(3) pp. 313–329.

Osmoregulation – an overview | ScienceDirect Topics (n.d.). [Online] [Accessed on 2nd December 2017] https://www.sciencedirect.com/topics/neuroscience/osmoregulation.

Regulation of Water Balance (n.d.). [Online] [Accessed on 5th December 2017] https://courses.washington.edu/conj/bess/water/water.htm.

Concepts in molecular biology – failures in meiosis which could resulting in chromosomal disease

Meiosis is a cell division process in which haploid gamete cells are produced in a diploid organism. A diploid organism is an organism which has homologous copies of each parent chromosomes within their cells. The parents of the offspring each donate a set of chromosomes which will then equal to two sets (Xu, 2006). Humans have 46 chromosomes within their diploid cells, compared to the 23 chromosomes which can be found within haploid cells. Haploid cells can be found within the gametes of diploid organisms, otherwise known as the sperm and ovum cells or reproductive cells. Haploid cells only have one set of chromosomes because when two parent haploid cells come together, they become fertilised. This means that the offspring will gain a complete set of chromosomes and become a diploid cell or diploid organism. (Kent, 2013).

Meiosis is an incredibly significant process for maintaining life itself. MacLennan (2015) states that meiosis is essential as it allows for the reduction of chromosomes within the gamete cells to maintain the correct number of chromosomes within the offspring after fertilisation. This is to reduce chromosomal disease and maintain genetic diversity within a species. Meiosis creates genetic diversity by recombining the chromosomes genetic material. Genetic variation is then increased further when the two parent gametes come together during fertilisation, there by creating an offspring with unique DNA combinations. (Humphryes and Hochwagen, 2014).

There are two consecutive divisions that take place during meiosis and the result of the processes of meiosis is the production of four haploid daughter cells. These cells that are produced are genetically different from their parent cells and the four cells that are produced are even genetically different from each other. This is because during meiosis, a pair of homologous chromosomes can exchange genetic material before being separated. (Kent, 2013). Meiosis firstly begins with Interphase. During interphase the chromosomes and organelles within a cell begin to duplicate. This results in each chromosome consisting of two daughter chromatids. (Simon et al., 2013). Interphase is also used to store energy as ATP (Adenosine Triphosphate) which can then later be used during the meiosis process. (Kent, 2013).

The process then moves on to Prophase 1. This stage consumes around 90% of the division time as it is an extremely complex stage. During Prophase 1 the chromosomes begin to condense, the nuclear envelope disintegrates, and the spindle apparatus starts to appear. (Kent, 2013). As the chromosomes begin to condense and coil up, proteins cause the homologous chromosomes to draw together and form pairs. The results of this are structures consisting of four chromatids. Within each structure, chromatids start to swap corresponding segments. This is also known as “crossing-over”. The process of crossing over means that the genetic information present is then rearranged. This is how the cells that are produced become genetically different from their parent cells, and each other. (Simon et al., 2013).

The next step is Metaphase 1. During this stage the homologous chromosome pairs begin to line up along the equator of the cell. Spindle microtubules from one pole attach to the centromere of one chromosome, and spindle microtubules from the other pole attach to the centromere of the chromosome’s homologous pair. The centromere does not divide so the sister chromatids remain together. (Kent, 2013).

The homologous chromosomes begin to separate and migrate towards the opposite poles in which they were attached to. (Kent, 2013).  The chromosomes are separated from their homologous partners, but the sister chromatids move as a single component. (Simon et al., 2013). This process is known as Anaphase 1.

The final stage of meiosis 1 is Telophase 1 and Cytokinesis. The chromosomes finally arrive at the poles of the cell. Once they arrive each pole consists of a set of haploid chromosomes, but the chromosomes are still doubled. Cytokinesis coincides with Telophase 1 and two haploid cells are produced. (Simon et al., 2013).

The process of Meiosis 2 results in the separation of the sister chromatids pairs that were separated from their homologous pairs in Meiosis 1. Meiosis 2 begins with a haploid cell that has not undertaken duplication of chromosomes during the Interphase period. This process is considerably similar to that of Meiosis 1. (Kent, 2013). A spindle forms during Prophase 2 which pushes the chromosomes into the centre of the cell. During Metaphase 2 the chromosomes align, with spindle microtubules from opposite poles attaching to the sister chromatids of each chromosome. The centromeres of the sister chromatids separate during Anaphase 2, and the sister chromatids of each pair begin to migrate towards the opposite poles. Finally, in Telophase 2 nuclei begin to form at the poles. Cytokinesis also occurs at this time, and the final result is four haploid daughter cells, each consisting of single chromosomes. (Simon et al., 2013).

The meiotic cell division process does not always run its course perfectly. When things go wrong during meiosis, the effects can be projected through chromosomal disease. Chromosomal disease can be fatal to an embryo. Qi et al, (2018) shows that during their study 42.95% of embryo miscarriage samples, out of 149 samples, all contained at least one chromosomal abnormality. Not all cases of chromosomal abnormalities are fatal, but there are a variety of problems that can occur. Errors in meiosis are referred to as nondisjunction. This means that during the meiotic cell division phase either the homologous chromosomes or sister chromatids failed to separate properly.  Turner Syndrome is a condition that affects around 1 in every 2,500 female babies. The condition means that these female babies are born with only one X chromosome. Normally females are born with two X chromosomes. Because these females are lacking the second X chromosome, their ovaries normally degenerate before they are even born. This means that the females can not undergo puberty. The condition can be treated to an extent by providing oestrogen as a hormone treatment, these would stimulate secondary sexual characteristics such as enlarged breasts, but because the females do not have functioning ovaries they still would not be able to produce offspring. Other characteristics of Turner Syndrome can include; increased risks of cardiovascular disease, kidney defects and hearing loss. (Audesirk et al., 2017)

Another common chromosomal abnormality is Trisomy 21, or Down Syndrome. Trisomy 21 is a condition where by the offspring has accumulated an extra copy of chromosome 21. Children that are born with Trisomy 21 usually have very distinctive physical abnormalities. Some of these can include; a smaller mouth that can only open partially to try and accommodate for a much larger tongue, weak muscle tone and distinctive eye shape. These characteristics also go along side some degree of cognitive impairment (depending on the person) and learning difficulties. Over the years there has been an improvement of health and life longevity for people who suffer from Trisomy 21, but this increase in life longevity has also been linked to the rise in people with Trisomy 21 suffering from Alzheimer’s disease as they get older. Chromosomal abnormalities such as Trisomy 21, occur during meiotic cell division. Abnormalities can occur due to increased maternal age (Prenatal screening for Trisomy 21 is usually advised for mothers over the age of 35) but this isn’t always the case as babies who suffer from this condition have been born to mothers under the age of 35. (Diamandopoulos and Green, 2018).

In conclusion meiosis is vital for life to continue and succeed. Without the success of meiosis every species of living diploid organism would eventually become extinct, as organisms would not be able to further reproduce as a result of haploid gamete cells not being produced. Gamete cell production is essential for the natural continuation of a species. Although meiosis is vital for reproduction, it is also essential for genetic variability. Genetic variability allows new organisms to be produced with a completely unique set of DNA. Without genetic variability the risk of chromosomal disease can be much higher. But, chromosomal disease can occur in any new offspring, even with genetic variability present within their parents. With this in mind, chromosomal disease should continue to be researched to understand more as to why failures in meiosis can arise.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

References

Audesirk, T., Audesirk, G. and Byers, B. E. (2017) Biology Life on Earth. Eleventh.

Diamandopoulos, K. and Green, J. (2018) ‘Down syndrome: An integrative review.’ Journal of Neonatal Nursing, February.

Humphryes, N. and Hochwagen, A. (2014) ‘A non-sister act: Recombination template choice during meiosis.’ Experimental Cell Research. (DNA DAMAGE AND REPAIR), 329(1) pp. 53–60.

Kent, M. (2013) Advanced Biology. 2nd ed.

MacLennan, M., Crichton, J. H., Playfoot, C. J. and Adams, I. R. (2015) ‘Oocyte development, meiosis and aneuploidy.’ Seminars in Cell & Developmental Biology. (Plasma membrane repair & Development and pathology of the gonad), 45, September, pp. 68–76.

Qi, H., Xuan, Z.-L., Du, Y., Cai, L.-R., Zhang, H., Wen, X.-H., Kong, X.-D., Yang, K., Mi, Y., Fu, X.-X., Cao, S.-B., Wang, J., Chen, C.-J. and Liang, J.-B. (2018) ‘High resolution global chromosomal aberrations from spontaneous miscarriages revealed by low coverage whole genome sequencing.’ European Journal of Obstetrics & Gynecology and Reproductive Biology, 224, May, pp. 21–28.

Simon, E. J., Dickey, J. L. and Reece, J. B. (2013) Campbell Essential Biology. Fifth.

Xu, J. (2006) ‘Extracting haplotypes from diploid organisms.’ Current issues in molecular biology, 8(2) p. 113.

Professional practice, Task D – data handling reflection

During first year at university I have already gained a respectable amount of knowledge through the subjects I have been taught but I have also gained a reasonable amount of applicable skills. The ability to self-reflect has been a very accountable skill that I have gained. Self-reflection gives me the opportunity to look back to the beginning of the year and see how far I have progressed. It also allows me to identify my weaknesses, making me aware of the areas that I need to improve on if I want to be able to better myself for second year and further.

Assessing the grade I achieved from the sector studies exam indicates that there are a variety of areas that I need to improve on in order to better my studies. Comparing the mock that was completed prior to the exam, to the exam itself, I can determine that I struggled with several maths questions. In both the mock exam and the official exam I failed to complete questions referring to graphs. Pareja-Lora et al., (2016) states that mock exams are incredibly useful not just for revision purposes but to highlight strengths and weaknesses to help aid student development. Mock exams give students the opportunity to reflect on their results and gain feedback from their teachers to help develop any weaker skills. With this in mind, I should have engaged with my teacher and asked if it could have been possible for me to go over the graph question and other questions I didn’t quite understand. This would have allowed me to advance my maths skills and may have allowed me to obtain a higher grade. This argument can be developed further by Zhang and Hyland (2018) who proclaim that students who actively ask for teacher feedback gain better understanding and retention of information within that subject.

Referring to the self-reflective task that was completed at the beginning of the year, I refer to use of Self Directed Learning being a useful tool for improving skills “One way of working on a classroom flaw or struggle is to undertake Self Directed Learning (SDL)”. I still stand by this statement as SDL is extremely beneficial to students, as it involves integrity to address weaker areas of study and enables students to take extra time to work on said areas through a variety of different activities. The engagement of SDL activities leads to improved educational results for students (Kastenmeier et al., 2018). SDL can also be considered a form of continuing professional development (CPD). CPD is the act of an individual taking control of their own learning and career development. This is usually done through the act of reflection and action. If an individual has highlighted areas of improvement, they will then take action and physically do something to improve their learning i.e. courses, conferences, outside reading, work experience etc. (Megginson and Whitaker, 2017) This argument is continued further by Wareing et al.,(2017) who suggests that the act of CPD is not only beneficial to the individual, but to anyone who is involved with said individuals work. This could include, working peers, customers, patients etc depending at the career within questions.

To conclude I aim to develop myself further through the suggestions listed above. Within development I may not see a progressive change in my grades and skills set as I move into my second year. Although, these skills are not just confined to grades within university. The can be carried with me throughout my career as their will always be room for improvement and developed in whatever career path I choose.

 

 

 

 

References

Kastenmeier, A. S., Redlich, P. N., Fihn, C., Treat, R., Chou, R., Homel, A. and Lewis, B. D. (2018) ‘Individual learning plans foster self-directed learning skills and contribute to improved educational outcomes in the surgery clerkship.’ The American Journal of Surgery, January.

Megginson, D. and Whitaker, V. (2017) Continuing Professional Development. Kogan Page Publishers.

Pareja-Lora, A., Calle-Martínez, C. and Rodríguez-Arancón, P. (2016) New perspectives on teaching and working with languages in the digital era. Research-publishing.net.

Wareing, A., Buissink, C., Harper, D., Gellert Olesen, M., Soto, M., Braico, S., Van Laer, P., Gremion, I. and Rainford, L. (2017) ‘Continuing professional development (CPD) in radiography: A collaborative European meta-ethnography literature review.’ Radiography. (Radiography Education), 23, September, pp. S58–S63.

Zhang, Z. (Victor) and Hyland, K. (2018) ‘Student engagement with teacher and automated feedback on L2 writing.’ Assessing Writing. (Special Issue: The comparability of paper-based and computer-based writing: Process and performance), 36, April, pp. 90–102.

Professional development plan

 

Professional Practice Task C – Professional development plan

Whilst I am currently still only within my first year of my Bioveterinary Science degree, I have begun to think about my future career or post graduate study after completion of my first degree. I have multiple interests that have arisen since starting the course. Writing a professional development plan allows me to break these interests down into achievable goals, outlining what specific skills I will need, and how I can better myself to attain these skills. Creating a plan for my future career gives me the ability to pin point areas within my study skills that may need to be improved on to allow me to accomplish the grades I will need to either apply for particular post graduate jobs, or move onto post graduate study such as; Veterinary medicine, Masters degree, etc.

One of my academic skills that I wish to advance is my revision strategies. Developing this skill will allow me to successfully complete my degree and give me the grades I need to move onto further study, such as Veterinary Medicine. I will need to attain a 1st  class degree in Bioveterinary Science to have the chance to apply for the Veterinary Medicine accelerated course. Reflecting on exam results from earlier on in the semester, I feel as though I could have accomplished better grades. I believe I would have retained much more information if my revision skills were better. I can make this goal achievable by making small changes to the way I study. Firstly, I can create flash cards after each lecture, rather than trying to make flash cards on all the lectures after they have all been completed. This way, the information will still be fresh in my memory and it will be less time consuming. I can measure my progression by taking note of how many flash cards I get correct each week. If I am not progressing with certain cards, I will know that I will need to put aside more time to revise those certain areas of the topic. Setting aside enough time for revision will also be beneficial. Doing small sessions every week will allow me to retain more, without getting stressed about time and how much content I have left to revise. These strategies will be helpful for both my Comparative Anatomy and Physiology module and my Concepts in Molecular Biology module.

Networking is also incredibly valuable when it comes to creating a career plan. As I am still unsure of the path I want to take after completing my degree, networking and work experience is a great way to engage with scientists within different fields and learn about their own careers. There are a variety of ways that I could make this possible. The Royal Society of Biology (RSB) organise many different conferences and careers fairs that are available to student members, such as myself. As a student member it would be possible for me to volunteer at any of the conferences that take my interests. To make this possible I would have to email the RSB and explain my current situation; where and what I’m studying, why I would like to volunteer, etc. If they allow me to volunteer, it would give me to opportunity to attend one of these conferences, whilst working. It would be useful for me to bring a small notebook and pen with me to take note of any contacts of tips that may be given to me. This way, if I gain the opportunity to get talking to different scientists at a conference I could ask them about their careers and what led them down that path and what skills and grades they needed to get to where they are. Networking during conferences is also a useful tool to gain work experience. Again, by talking with scientists within different fields, it gives me the chance to ask whether it would be possible for me to gain work experience with them, or if they know of any companies/institutions that would be willing to take on students looking for work experience. Work experience is extremely helpful when creating a professional CV when looking for post graduate jobs. Having relevant work experience on my CV could put me in a different category to other candidates as it shows that I not only have the grades needed, but also relevant practical skills, experience and enthusiasm. Work experience would allow me to explore all my different interests and see what the work within that field is like. Exploring different jobs and working environments through work experience could be extremely helpful for me to put together a well thought out decision about post graduate jobs or study as it should allow me to delve into my interests and see which is the one I am most passionate about and would like to develop a career in.

Laboratory Report

 

Abstract

DNA fingerprint matching using PCR is an accurate and affective way of identifying or matching DNA to an available sample. It is used widely throughout forensic science and is spoke about very highly within further research. Throughout this report the positives of PCR are discussed, along with the use of equipment and how this method is still effective, even when not accomplished completely accurately.

 

Introduction

The purpose of this experiment was to identify which suspects DNA, out of the four samples provided, matched the DNA that was given as DNA found at a scene. The DNA matching was completed using Polymerase Chain Reaction (PCR). The DNA used within this experiment was provided in a DNA fingerprinting experiment kit, created by Edvotek (EDVOTEK, 2017). However, DNA matching using PCR is frequently used by forensic professionals to determine DNA profiling of suspects using minute amounts of DNA which has been extracted from a crime scene. (Sinelnikov and Reich, 2017). Cavanaugh and Bathrick (2018) argue that PCR amplification is one of the most effective forms of DNA matching within forensic science. PCR is found to be more effective than other methods as DNA samples can be directly added to an amplification reaction, rather than being exposed to DNA extraction, purification or quantification. All of which can damage or contaminate DNA samples. This method also allows maximum amounts of DNA to be extracted, allowing for less error when matching DNA fingerprints. This is a vital advantage when working within forensic science.

Materials

  • DNA samples (provided by EDVOTEK)
  • 5 PCR tubes
  • Primer mix
  • PCR Edvobead
  • Centrifuge
  • (50x) Buffer
  • Distilled water
  • Flask
  • Microwave
  • 7x7cm casting tray – with rubber ends and well templates
  • Electrophoresis chamber
  • Timer

Method

Firstly, we began by labelling 5 PCR tubes to ensure that the experiment was completely accurate. The tubes were labelled; Crime scene (CS), suspect 1 (S1), suspect 2 (S2) suspect 3 (S3) and suspect 4 (S4). To prepare each individual PCR reaction, each tube was filled with 20 micro litres of the primer mix provided, then 5 micro litres of the crime scene DNA (for the PCR tube labelled CS) and finally one PCR Edvobead. This was repeated for each tube, but the appropriate DNA was added to the appropriately labelled tube. Each tube was then gently shaken to mix the solution inside, and to ensure that the Edvobead was fully dissolved. Next, the samples were placed inside the centrifuge to spin and separate the DNA from the rest of the solution, making it easy to collect from the bottom of the tube. The DNA was then amplified using PCR. The PCR cycle started with initial denaturation starting at 94 degrees Celsius for 3 minutes. After, the cycle continued with conditions of 94 degrees Celsius for 30 seconds, then 55 degrees Celsius for 65 seconds and then 72 degrees Celsius for 30 seconds. This cycle was repeated 30 times. Before beginning electrophoresis, 5 micro litres of 10x gel loading solution was added to each tube.

The next step was to dilute 0.5ml of concentrated (50x) buffer with 24.5ml of distilled water into a flask. 0.25g was then added to the solution to create a total volume of 25ml. The agarose powder was then dissolved in the solution by microwaving it on high for 1 minute. The flask was taken out of the microwave and swirled to see if the agarose had dissolved. The flask continued to be heated for short 15 second bursts until the liquid was completely transparent – indicating that the agarose had been dissolved. The was set aside to cool down, whilst waiting, the rubber end caps were placed at the ends of 7x7cm the gel-casting tray that the gel would be placed in. The well template, or comb, was then clipping into the tray, ready for the solution to be poured in. Once the flask containing the solution had cooled down enough so that it could be touched without gloves, the agarose solution was poured into the gel-casting tray. The gel was left for over 20 minutes to allow enough time for setting. Once the gel was completely set the rubber end caps were removed, along with the comb. The comb had to be removed extremely carefully to ensure that none of the well moulds were damaged.

The tray containing the gel was then placed inside the electrophoresis chamber. 1x electrophoresis buffer was then poured into the chamber, until the tray was completely submerged. Lane 1 of the wells was filled in with the ladder sample. Then following that each DNA sample that was prepared earlier (CS,S1,S2,S3,S4) was placed in an individual well within the gel, in that order. The safety cover was then clipped onto the chamber, and the appropriate leads were attached to the appropriate power source (Red – Red, Black – Black). The electrophoresis chamber was then turned on for approximately 55 minutes. After the electrophoresis was complete, the gel trays were removed from the chambers, and the gel was then removed from the tray. The gels were then taken to a different lab where they were scanned by a technician to produce an image of the results.

(EDVOTEK, 2017)

Results

L S4 S3 S2 S1 CS L

(Essential Laboratory Techniques : Crime Scene Gel, n.d.)

The image above is the photo that was produced after the gel had been scanned by the technician. From order of right to left, the wells read; Ladder marker, crime scene, suspect 1, suspect 2, suspect 3, suspect 4 and the final ladder marker. As shown by this image the suspect that was the closest match to the crime scene DNA was suspect 3, as indicated by the DNA highlighted within the wells.

Discussion

From the results shown in the image provided, it can be seen quite clearly that the wells at the top of the image are more intact and more obvious to see. But towards the bottom of the image it appears the wells become less distinct. As discussed previously, the agarose gel solutions were set in gel casting trays size 7x7cm, and the electrophoresis chamber was turned on for around 55 minutes. From the results shown we can see that the wells almost taper off, towards the end of the image. As shown from the image below it is quite clear that all the wells are intact and obvious to see when the gel was scanned for an image.

(Zhang et al., 2015)

This could suggest that the agarose gel became damaged during the experiment because as the DNA was migrating during the electrophoresis (DNA migrates towards the red [positive] electrode) the gel may have become damaged if the chamber was too small and if the current was too strong. If the DNA didn’t have enough room to migrate properly the larger sections of DNA could have damaged the wells whilst travelling, creating a less clear picture. With this in mind, the electrophoresis chamber may also have been left on for too long, although the manufacturer guides were followed correctly.

Although the gel may have been damaged during the experiment, it is still clear from the results which suspect DNA matched the DNA that was found at the crime scene. This gives indication that although the experiment may not have been completed 100% correctly, it still shows how accurate PCR can be, giving and indication of why it is so popular and widely used within forensic science and other sciences. (Pounder et al., 2005)

Conclusion

In conclusion, the experiment was successful in determining which suspect DNA matched that of the crime scene DNA. DNA fingerprint recognition using PCR has been proven to be extremely successful and accurate. The method produces clear imagery and evidence, even if the process of electrophoresis isn’t always correct. This shows the benefits of PCR and how determining and matching DNA fingerprints has developed.

References

Cavanaugh, S. E. and Bathrick, A. S. (2018) ‘Direct PCR amplification of forensic touch and other challenging DNA samples: A review.’ Forensic Science International: Genetics, 32(Supplement C) pp. 40–49.

EDVOTEK (2017) ‘DNA fingerprinting using PCR.’

Essential Laboratory Techniques : Crime Scene Gel (n.d.). [Online] [Accessed on 9th January 2018] http://moodle.writtle.ac.uk/mod/resource/view.php?id=105232.

Pounder, J. I., Williams, S., Hansen, D., Healy, M., Reece, K. and Woods, G. L. (2005) ‘Repetitive-Sequence-PCR-Based DNA Fingerprinting Using the DiversiLab System for Identification of Commonly Encountered Dermatophytes.’ Journal of Clinical Microbiology, 43(5) pp. 2141–2147.

Sinelnikov, A. and Reich, K. (2017) ‘Materials and methods that allow fingerprint analysis and DNA profiling from the same latent evidence.’ Forensic Science International: Genetics Supplement Series, 6(Supplement C) pp. e40–e42.

Zhang, Y., Suehiro, Y., Shindo, Y., Sakai, K., Hazama, S., Higaki, S., Sakaida, I., Oka, M. and Yamasaki, T. (2015) ‘Long-fragment DNA as a potential marker for stool-based detection of colorectal cancer.’ Oncology Letters, 9(1) pp. 454–458.

Task D – Reflective piece

Fundamentals in Bioveterinary Science – Task D

Self-reflection is extremely important to help with skill development, especially as a student. It allows you to refer back to what you once thought may have been a flaw or a weakness, and gives you the ability to see how far you have developed, or to discover what skills may still need improving.

Within this stint of the semester I feel as though I have gained more confidence within our maths and chemistry lectures than I did at the beginning. I now feel a lot more comfortable with participating and raising my hand to speak in class, even if I am wrong. At the start of the year I felt anxious to answer a question as I knew chemistry and maths were my weaker subjects. Murray and Lang (1997) discuss that there is a definite link between student participation and gain in knowledge retainment and problem-solving skills. This is beneficial to me as the gain in knowledge and understanding, through active participation, can be associated with an increase in confidence within that subject.

Referring back to my previous reflective writing that was completed at the beginning of the year I spoke about feeling more comfortable working with students who are of the same academic capability as myself; “I feel as though the lesson would have been more productive if the class was separated into different skill levels”. Although I previously thought that surrounding myself with those who are of the same capability as me would be more productive when it came to understanding lesson content, I have now deemed this to be not entirely true. I have found it easier to turn to a student within my class, who may have come from a chemistry and maths background, as the majority of the time they pass on useful tips or ideas to help make content easier to understand. Linchevski and Kutscher (1998) state that studies show a significant difference in achievement between less able students who were placed in mixed ability teaching groups compared to those within same ability teaching groups. This theory is also backed up by further studies which suggest that students within mixed ability groupings find that they can elaborate on the subject matter on their own, rather than as a collective. (Saleh et al., 2005).

Although I have improved on my chemistry and maths I do still find it a struggle at times. Personally, I find practical lessons quit daunting as I haven’t been exposed to many chemistry practical lessons prior to starting university. In my previous piece of reflective writing I spoke about the use of Self Directed Learning (SDL) being useful for improving classroom flaws. SDL is the act of an individual taking it upon themselves to carry out actions that will be beneficial to improving their learning. (Rothwell and Sensenig, 1999). I feel as though the best way to improve on my practical work is to simply just practice. After a lesson I could ask the lecturer to explain the purpose of any equipment I am unsure of, or go over a portion of the lesson that I didn’t quit understand. (Beasley, 1985). Also, when thinking about the benefits of mixed ability groupings, as mentioned previously, it could also be advantageous for me to ask to work with a student who may be more confident with chemistry practical’s.

During this reflective piece it has been enlightening to see the developments I have made within chemistry and maths, in a relatively short amount of time. It is encouraging to known that I can and have improved within my least favoured subjects. Although there are still flaws and a lot of work will have to be done so as I can continue to see these developments, I am confident that with the help from my lecturers and my peers I can continue to better myself.

 

 

 

 

References

Murray, H. G. and Lang, M. (1997) ‘Does classroom participation improve student learning?’ https://www.stlhe.ca/wp-content/uploads/2011/07/Does-Classroom-Participation-Improve-Student-Learning.pdf

Beasley, W. (1985) ‘Improving student laboratory performance: How much practice makes perfect?’ Science Education, 69(4) pp. 567–576.

Linchevski, L. and Kutscher, B. (1998) ‘Tell Me with Whom You’re Learning, and I’ll Tell You How Much You’ve Learned: Mixed-Ability versus Same-Ability Grouping in Mathematics.’ Journal for Research in Mathematics Education, 29(5) pp. 533–554.

Rothwell, W. J. and Sensenig, K. J. (1999) The Sourcebook for Self-directed Learning. Human Resource Development.

Saleh, M., Lazonder, A. W. and Jong, T. D. (2005) ‘Effects of within-class ability grouping on social interaction, achievement, and motivation.’ Instructional Science, 33(2) pp. 105–119.

Animal health industry analysis

During this report the topic of CCTV recording becoming mandatory in all slaughterhouses in England will be discussed. The Environment Secretary Michael Gove has proclaimed that the use of CCTV cameras within slaughterhouses will reassure consumers that abattoir workers are delivering the highest standard of animal welfare. The Food Standards Agency (FSA) are also highly in favour of the new legislation as they claim to have a zero-tolerance approach to any breaches that are made against animal welfare. (Mandatory CCTV in all slaughterhouses under new animal welfare plans – GOV.UK, n.d.) This report will also evaluate whether the new legislation will improve animal welfare and the effects it will have on the meat industry from a business point of view.

The consultation on CCTV in slaughterhouses started on 11th August 2017 and was set to run for the following 6 weeks after that date. The Environment secretary Michael Gove has stated that by putting these measures in place, it will demonstrate further to consumers throughout the globe that UK meat production is of the very highest standard. Qualified vets would be responsible for monitoring and evaluating the footage to determine the quality of the welfare given to the animals. (Mandatory CCTV in all slaughterhouses under new animal welfare plans – GOV.UK, n.d.) Harper and Makatouni  (2002) claim that results taken from focus group discussions indicate that consumers use animal welfare as an indicator of the quality, safety and healthiness of what they are buying, with most consumers being drawn towards organic or free range foods as its thought welfare standards of these products would be better. With this in mind, the new CCTV legislation that is being considered by the government could potentially improve animal welfare as well as generating more money for the meat industry. If the public are reassured that all meat that is slaughtered in the UK is up to the highest welfare standard, they may feel more obliged to buy meat from smaller producers or supermarkets rather than organic/free range producers. If improved welfare standards are what’s going to generate more money for the meat industry than producers will feel more obliged to abide by the law and give their animals, the highest quality welfare.

In 2004 the government produced a Strategy for Animal Health and Welfare for Great Britain. The vision of the strategy was to improve and sustain animal welfare for the following 10 years. One of the statements of the strategy specified “Consumers value the confidence they have in food produced safely from healthy animals that are well cared for. Consumers and retailers accept that higher standards of animal health and welfare are not cost free.” (Council, 2009). Statistics produced by the Agriculture and Horticulture Development Board show that the average farm and retail price of beef, lamb and pork has been increasing since August 2016 – October 2017. (AHDB Beef and Lamb, 2017)  This may be due to a demand in higher welfare standards and quality of product. The new CCTV legislation that is being discussed may cost the industry and the tax payer more money. Referring back to the strategy produced in 2004, it also states “The costs of livestock health and welfare are appropriately balanced between industry and the taxpayer.” (Council, 2009) The cost of providing all slaughterhouses in England with CCTV cameras, as well as paying qualified vets to review the footage and determine the quality of the animal’s welfare may work against the industry from a financial point of view. If the cost of improving welfare becomes too high, consumers may stop indulging in the meat industry or purchase meat that has been imported if it means it will be cheaper.

To conclude, the introduction of CCTV into slaughterhouses in England could potentially improve the welfare of animals as the behaviour of abattoir workers would be constantly reviewed by qualified vets. This would potentially stop workers from breaching any animal welfare laws that they are expected to abide by. On the other side of this argument, the new legislation that is being considered could become very costly to the industry and potentially the tax payer. If the price of meat that is slaughtered in the UK becomes too exclusive, consumers may be obligated to purchase cheaper meat that has been imported into the country or stop contributing to the meat industry all together. If the CCTV legislation is to be put into place permanently the government will need to work out a way to make it cost effective whilst still improving overall animal welfare.

 

References

AHDB Beef and Lamb (2017) ‘Farm to retail price spread October 2017.’

Council, F. A. W. (2009) Farm animal welfare in Great Britain: Past, present and future. Farm Animal Welfare Council.

Harper, G. C. and Makatouni, A. (2002) ‘Consumer perception of organic food production and farm animal welfare.’ British Food Journal, 104(3/4/5) pp. 287–299.

Mandatory CCTV in all slaughterhouses under new animal welfare plans – GOV.UK (n.d.). [Online] [Accessed on 15th November 2017] https://www.gov.uk/government/news/mandatory-cctv-in-all-slaughterhouses-under-new-animal-welfare-plans.