Day 1 began with the task of extracting the DNA from the fruit fly larva. This lengthily process included squishing the larva in an Eppendorf using a pestle. We then added the pre-lysis buffer, which created the right conditions to continue. Then we added ethanol which adjusted the DNA binding conditions. We then bound the DNA sample to the column and washed and discarded the waste. The process was completed repetitively. Lastly, using the buffer we removed the DNA.
RESULT: The result from day 1 included the successful distraction of DNA, resulting in the isolation of the DNA from the specimen.
Day 2 was focused upon using the PCR (polymerase chain reaction) machine to isolate the DNA. Then, we used Gel-electrophoresis which separates the fragments according to their length/size. By using the PCR method, we were able to obtain enough DNA to allow it to be analysed. We completed the method of copying the barcoding region. Firstly we added the primer to the 5 HIP tubes. The primer attaches to a complementing sequence, and each one will stick to a different point of the gene. Then we added the “master mix”. The master mix was made up of nucleotides, Taq DNA polymerase and a buffer. Each of these ingredients have individual jobs, the Nucleotides (G,T,A,C) builds off the primer to create a copy of the DNA pair. The Taq DNA polymerase attaches to the primer and then runs along the sequence and attaches the nucleotides. The Buffer makes the conditions right for the polymerase to work. Lastly, we added our isolated genomic DNA solution. This is the template of the original sequence that gets copied. After combining all the elements we then transferred the tubes to the PCR machine which then ran for 2 hours.
We then moved onto the stage of Gel- electrophoresis. We did this by pouring a liquid gel substance into the required space of the tank. Once the gel was set, we then poured the buffer solution into the gel tank. Our last step was adding the DNA samples into the individual wells. We then placed the lid on the gel tank, and ran the gel on 100 volts for 15 minutes. Since the gel contained a fluorescent dye which binds to DNA, we then placed the gel under the UV light so we could examine the DNA results.
RESULT: Day 2 resulted in using PCR to copy and multiply the DNA sample into millions of copies. After copying the sample, we successfully used Gel-electrophoresis and separated the individual DNA fragments according to their size. From the gel-electrophoresis results, we were able to identify that black, blue and green had the most DNA. These colours worked most effectively, as they resulted in the largest amount DNA.
Day 3 consisted of 3 rotations. These rotations included:
Looking at fly genetics in which we visually discovered the differences between the wild flies and the mutated flies. This was discovered by looking at 7 different flies that had mutations and comparing them to the ‘controll’ fly. We used a microscope to be able to have a magnified view of the fly. We also learned to record and identify the punnet squares which allowed us gain a better understanding of how phenotypes and genotypes are determined through genetics.
We used an electron microscope to visualise the mutations of the ‘controlled’ version of a fly, and the mutated fly. We discovered that the mutated fly evidentially had different characteristics and tumour like bulges on the body. After discovering these differences, we zoomed in, adjusted focus on the computer and screenshotted all areas of the fly that looked different to the normal fly.
We looked at bioinformatics, as well as proteins and created a protein model, using amino acids. We examined some ‘RNA’ of proteins and compared them to the DNA sequence. After creating our own protein models, learned which amino acids are positively charged, negatively charged and which are attracted and repelled from one another. After creating our model, we deciphered both the RNA and DNA sequence of the protein.
RESULT – From the rotations of day 3, we discovered the mutations in the fly which we could visually see. We figured out the punnet squares and how they work. We learned to use the electron microscope to identify the mutations that are harder to see on a stereo scopes. Lastly, we learnt what RNA was, the different types of amino acids and which of them are positively and negatively charged.
Day 4 consisted of finishing off our final rotation and then preparing our blog and preparation. We learnt tips and tricks about designing, preparing and finalizing our team’s blog. We then joined forces with the other 3 groups, and we all worked on creating our blogs, for final presentation. After our lunch break, as a collective, we got the opportunity to go across, and explore the Peter McCullum cancer research facility. This tour was extremely rewarding, as it majorly contributed to the knowledge and understanding of the hippo gene, and the fly mutations we have been researching and experimenting on. In the first ‘rotation’ we went into a dark room, and visualized the tissue cell of the wild type fly. The tissue sample was displayed in different colour forms, on a screen which has been connected to the microscope. The next rotation included looking at cells from a microscope. These cells were being copied and mass created, to be made into samples which were the cells of the wild type and two different mutations. Our guide showed our group how to grow and care for these cells. Rotation 3 consisted of exploring the differences of appearance in 2 types of mutated flies, comparing their physical features to the ‘controlled’ type. We learnt about the life cycle of the fly, and the process of triggering the tumours. We looked at the larva under the microscope and looked at the mutations that were inherited by the experimental flies. Our guide told us about the importance and method of growing and modifying these flies for examination. Our last rotation consisted of learning about the advanced new way of experimenting on liver cancer, using zebra fish.
RESULT – On day 4, our group successfully broadened our understanding and knowledge of the cells, mutations, tumours and life cycles of the wild type fly. We enjoyed the tour of the centre, and took in lots of the technology and new things that were taught to us.
WHAT WE LEARNT ON OUR FINAL DAY!
During the morning of day 5, we learnt the process of sequencing DNA. The process is very similar to PCR (polymerase chain reaction). However, instead of adding regular nucleotides, fluorescent blockers are added, limiting the completion of the strand. The DNA then goes through Gel-electrophoresis where a laser converts the fluorescent colour into a DNA sequence.
Jordan – Throughout this process, I have gained an extensive understanding about the extraction, examination and sequencing of DNA. I enjoyed learning about the Hippo gene, and how it contributes to discovering more about cancer. I found the tour of the institution fascinating and learnt much more about the flies lifestyle/life cycle, mutation and how to detect tumours/alterations.
Emily - Throughout my experience at GTAC I have learnt a lot, and have gained a further understanding of the Hippo gene. I have enjoyed learning about isolating the DNA from the larva, learning about the Polymerase Chain Reaction (PCR), Gel Electrophoresis and looking at sequencing reactions. I also enjoyed completing the activities and being able to learn about the proteins, punnet squares that involved genotypes and phenotypes and lastly looking at the physical differences between the wild type fly and our hippo mutated fly. My favourite part about this experience was going across to the Peter MacCallum Cancer Centre and looking at the different jobs, as well as meeting the people who were willing to kindly share with us their passion and allow us to learn about their pathway and findings with the Hippo Gene.
Heba - My experience at GTAC’s Work experience was at the very least educational and inspiring. From lacking in interest and bore to now actually finding it somewhat fascinating, innovative and having an interest in pursuing it as a VCE subject. Out of the many valuable and sometimes challenging activities spaced throughout the week I favoured the punnet square and looking at different fly mutations in comparison to wild fruit flies. To me the comparison in physical features seemed interesting, but then also capturing this on a camera seemed to spike up my favour.
Krista - This week at GTAC has been great. I had been to GTAC before so I knew what to expect. I was excited to meet new people and learn new things. I was very happy with the amount of practical work that was performed, I was also happy with how much I expanded my knowledge. My favourite part about this week would have to be learning about PCR (polymerase chain reaction) and the steps that it takes to copy millions of strands of DNA.
Mikayla - Coming into this week I had no idea what to expect, but I was pleasantly supprised by the complexity and excitement of the hippo gene. I learnt a myriad of different things this week, from the process of collecting and isolating DNA to PCR and Gel Electrophoresis. I also learnt about proteins, the different amino acids they are made up of and how one change in a sequence letter can be responsible for a new amino acid being produced. My favourite activity however, would have to be examining different fly mutations under the microscopes and comparing them to the Wild Type. I am so greatful for the opportunity provided by GTAC and would encourage others to be apart of this amazing experience.
Roman - When I got here I was introduced to all of the GTAC employees and we started on a DNA extraction, then the next day we put the DNA into the PCR machine and then put it into gel to see what DNA would be researchable. On the third day we looked at hippo mutant flies and regular flies under a microscope and learnt how the genes would be passed on. And on Thursday we went on a site tour and look at how they researched flies and zebra fish. And finally today how they learn the DNA sequence