GTAC Girls (2019)
GTAC Girls (2019)
Setting up a PCR
Lexie: 22/07/2019 11:30AM - 12:45PM
A PCR or Polymerase Chain Reaction is a common method of replicating specific segments of DNA. Through the PCR method, we aimed to generate millions to billions more copies of our ‘special’ genes in vitro (In a test tube and machine rather than a living organism).
Into a test tube, we pipetted a combination of water, Master Mix (MM), Forward Primer (F), Reverse Primer (R) & Template DNA (T). The MM contains a concentrated solution of Taq Polymerase, named after the heat-loving bacteria (Thermo aquaticus) from which the enzymes were isolated from.
Our group learned how the primers bind to opposite strands of the DNA, connecting to one another via complementary base pairing. Both the F and R components of the solution contain primers (short regions of nucleotides) which form a starting point for the DNA when bound to the template strand (T).
After adding the primer, we then heated the DNA using the thermocycler in the initial step Denaturation- heated first to 98°C, which breaks up the DNA strands as the hydrogen bonds that hold them together weaken. This process provides the single-stranded template for the next step.
This is known as Annealing- cooling the PCR down to 55°C, so that the primers can bind to the complementary sequences on the single-stranded template DNA.
The last step was called Extension- where the machine heated up again to 72°C, here the Taq polymerase extends the primers by adding the rest of the complementary bases (the G’s, T’s, A’s and C’s) completing the whole strand.
All three steps cycle through 25 times, in order to replicate as many copies as possible.
Prepping for the bacterial transformation
Sandy: 22/07/2019 1:45PM —2:30PM
Now that we had completed the PCR process, we had to make the bacteria competent in order for it to take up a plasmid. First, we had to have a suitable vector, in which a plasmid, as well as the gene for the antibody, was extracted from the bacteria and cut with restriction enzymes. Then when added together, made the recombinant plasmid - it now contained the antibiotic-resistant gene as well - specifically to resist ampicillin (an antibiotic similar to penicillin). We also learned that bacteria have a relatively negative charge - the same goes for DNA, which meant they would repel each other - so we had to introduce CaCl2 or Calcium Chloride so that the bacteria may be more “attractive” for the DNA. The bacteria was eventually ready to move on to begin the process of bacterial transformation.
Mary: 22/07/2019 2:30PM —3:30PM
The process of bacterial transplanting took place directly after we transformed the bacteria. The main aim of this step is to grow the bacteria E.coli for further analysis. Our apparatus for this procedure involved pipettes and four agar plates.
We pipetted 100 microliters of the solutions: -pREC, PCR, +pRECcut and +pREC onto the labelled agar plates. The base of the plates consists of a jelly-like substance, which is the fluid culture and the agar. This fluid culture guaranteed that the bacteria would quickly multiply under hot conditions in the incubator that simulated the heat of the human body - at about 37. Since the purpose of this experiment is to extract an antibody that targets inflammation in the human body, the bacteria would have to thrive in that environment simulated in the incubator.
After we pipetted 100 microliters of each of the solutions, we used a spreader to spread the culture around the plate, ensuring we spread every centimetre on the plate so that the bacteria receive maximum potential to grow. We sealed off the edges of the plates with parafilm in order to prevent any moisture loss and breathability. By completing this procedure, we are giving the bacteria two new properties - the ability to survive the ampicillin attack and incorporating the antibody gene into the genome.
We hypothesised that since the pREC+ plasmid is circular shaped, the bacteria would survive since it is a whole. The bacteria were able to make a copy of itself and survive the ampicillin. Because it is circular, it can produce the ampicillin resistance protein. This is also the evidence that we have added the antibody gene into the solution.
Preparing the Gel electrophoresis
Sandy: 23/07/2019 9:45AM-10:30AM
During our morning session, we also learned about how to transcribe the template DNA nucleotide sequence into mRNA nucleotide sequence and in the isolation of the antibody protein. We used a column to collect the flowthrough, which is essentially the “waste” products, in order to isolate the protein. We then used the electrophoresis machine, which separates macromolecules like DNA, RNA and proteins. DNA fragments are separated according to their size.
Protein Extraction and Isolation
Ha: 24/07/2019 11:30AM-1:00PM
We started by exploring the protein structure and then we extracted the protein and isolated it, and finished by using the gel electrophoresis. We used an e-gel system to compare protein structures in the ladder, in the first well we put molecular weight standards, in well three we put culture supernatant, in well four we put the flow-through (everything -ab) and in wells five, six and seven we put elute 1, 2 and 3 (ab)
Protein Gel Electrophoresis
Ha: 24/07/2019 2:00pm-3:30pm
After we have the entire sample, we will place it into the heat block up to 70 degrees in 10 minutes in this process the heat will help to break down the antibody. Next step is to put that solution into the microfuge about 10 seconds to mix it well. Now is a time to use electrophoresis machine, to this part we have to be very careful and slow to put all the sample into lanes, each lane will contain 15 microliters. Load 15 microliter of molecular weight ladder into Lane 1 and load 15 microliters of our sample into wells lanes 3 to7. When finish filling up all the lanes we start to run the gel at 200V for 40 minutes, at this stage the machine will help us to separate the charge of the molecules such as protein and cells according to their response in electric current. Continuing that we will take the gel out of the machine, put it into black liquid, and leave it overnight. The next day all the solution in the lanes all appear in the form of ladder, in each level of the ladder of solution have a dark blue in it means that solution have a good or strong antibodies in it.
- Hydrophilic ( attracted to water)
- Hydrophobic ( repelled from water)
- Acidic (negatively charged)
- Basic (positively charged)
One example of an amino acid is Asparagine. Asparagine is hydrophilic (or water loving). It has two carbon atoms, one nitrogen atom and an oxygen atom.
A Codon Wheel (as seen above) is a circular wheel used to identify the amino acid created from an RNA strand. DNA strands are composed of four base letters: A, C, G, and T.
The T is changed to a U when the DNA is transcripted into a strand of RNA. As the cell destroys RNA when it is roll is completed, but also does not want to destroy DNA, it has to be able to identify the two separately. Therefore, if RNA had a T instead of a U, DNA and RNA would look identical and the cell could possibly destroy DNA by accident. Therefore, the U’s purpose is to distinguish the different strands.
ELISA Stage 1
Mary: 25/07/2019 9:45AM-10:30AM
On this morning, we prepared our extracted antibodies for Enzyme-linked Immunosorbent Assay (ELISA). The aim of this procedure is to detect the binding affinity of the purified antibody. The first step of this procedure involves coating the base of our plastic wells with the protein of choice - the antigen. We started by setting up our benches with the required apparatus (insert photos). On our bench, our Mentor Duncan handed us four bottles, two of which contained our extracted primary and secondary antibodies and the other two containing primary and secondary antibodies provided by the CSL scientists. The reason why we used pH 5 is to simulate the properties inside the mammalian cell once the bacteria had been engulfed by a white blood cell and pH 7 is used to simulate the properties outside the cell. We used tested it under 37 degrees, which is the average temperature of the human body.
The list of steps required for ELISA are as followed:
- Coat the plastic wells with the protein of choice - (antigen)
- Add the purified antibody into the wells
- Add the secondary antibody with the enzyme attached
- Add the substrate for the enzyme
- Add the acid to halt the reaction
Before we had begun any work, Duncan ran through the protocol procedures we adhered to. We had to prepare a test group and a control group for the experiments. A serial dilution plate for both the test group and the control group would be split in half and contain the two different pH conditions. After that, antibodies 1 and 2 would be pipetted into each pH. After mixing the pH solutions with the antibodies, we were to transfer the solutions onto the ELISA plate for the wash - which is the test used to test for the binding ability of the antibodies.
Thanks to Daria at CSL, the wells were already coated with our unknown protein of interest specific to the purified antibody. However, these wells were blocked with a milk solution so that the proteins would not be able to escape. Inside the wells went each of the pH buffers. We were able to use the multichannel pipettes to transfer 250 microliters of pH5 or pH7 buffer into column 1. Furthermore, we transferred into columns 2-4 200 microliters of pH5 or pH7 buffer. Using the pipette again, we added 2 microliters of our purified antibody 1 (from columns GH and CD) and antibody 2 (from columns EF and AB) to column 1. To guarantee that the microorganisms were evenly displaced around the column, we gently mixed the solutions from column 1. 50 microliters from column 1 was transferred to column 2. Once again, the solution in column 2 was mixed. We repeated these steps again for columns 3 and 4.
Photo of the milk buffing solution in the wells.
We had to remove the milk buffing solution by vigorously tapping the coated plates onto dry paper towels. Our solution from column 4 on the serial dilution plates were transported to column 4 of the serial dilution plates to column 4 of the ELISA plates (both Test and Control groups). The steps for the procedure were repeated again for columns 3, 2 and 1 in that order. We used the same tips since we did not cross contaminate any solutions.
The plates were then sealed and left to incubate for 1.5 hours. This is to prepare for the secondary antibody.
We learnt that a positive reaction meant that the purified antibody would be able to bind to the protein at the base of the well. If nothing bonded to the antigens, the result would be a negative reaction since it is not our desired result. Furthermore, we learnt that the secondary antibody contained a special element, which helped it to bond tightly to the primary antibody.
After that, we added the secondary antibody with the enzyme attached. The secondary antibody contained a special element, which enabled it to bind to the primary antibody. It also contained an enzyme, which would change colour once the substrate is added to the solution.
ELISA Stage 2: Washing
Mary: 24/07/2019 12:00AM-1:00PM
To prepare our specimens for a wash, we first pipetted 2 microliters of the primary antibody and the secondary antibody in their correct pH suitable for the cells - pH 5 is to ensure that it matches the properties of the liquid inside the cell. pH 7 is to replicate the properties of the exterior of the cell.
In order to begin washing our cultures to test the effectiveness of our extracted antibodies, we took our wells to the automatic plate washer. The apparatus used a special buffer to wash out the ineffective antibodies, which did not bind to the protein at the base of the well. The purpose of this stage is to wash out any antibodies, which did not bond to the antigen at the base of the well. In this instance, this would mean that antibodies would be ineffective and would produce a negative result.
ELISA STAGE 3: Results
Our results have shown with the antibody test that there was potentially a pipetting error, which gave us corrupted results. The test tray of the antibody that Glen (from CSL) prepared for us worked and showed that we had the highest rate of enzymes in Ph7. In order to test the results of our antibody, we used a special liquid called an enzyme - a substance produced by a living organism, which behaves as a catalyst. The liquid - called TMB suddenly turned a deep blue, which converted the substrate from a clear molecule to a blue molecule. 100 microliters of TMB went into our pH solutions containing our antibodies and proteins. We left the enzyme to do its work and react with our solutions for 5 minutes. Since the reaction would continue to react unless stopped, we had to pipette 50 microliters of phosphoric acid to each well. The substrate instantaneously turned yellow which marked the end of the reaction.
Our ELISA results showed that the Glen’s antibodies were more effective than our antibodies. This is shown by the shade of yellow present since phosphoric acid was added. The deeper the shade of yellow, the more effective the antibody.
Britt: I was inspired to go to GTAC because of a sudden interest in science I have had this year; As soon as I heard about it I knew I wanted to go. I am so lucky to have been chosen to be a part of this amazing experience and very grateful for everyone who has made my week so helpful towards my passion for science. The whole experience has motivated me to aim even higher in science in schooling and has opened my mind to many new ideas. I thank all the GTAC team and scientists who came in and answered all my questions, I have learnt so much in such a small amount of time, and I cannot wait to share the information with other people who also have an interest in science!
Ha: I went to GTAC for work experience because it helped me to skip school and hang out with friends in the city but when I participate at GTAC, I kind of surprise that every experiment and assignment we do is very different to our school, all the equipment in the lab that I never heard or touched before, it just like one huge step for me to the real life and our lovely mentors always helping us and tell what to do. I sure with you guys that when I come back to school I will tell all the awesome things in the lab to my friends and gonna make them jealous.
Heidi: I absolutely loved the experience I had this week at GTAC. I learnt so much in a short amount of time. I had high expectations for this program but never actually thought it would be this interesting and valuable. It has doubled my interest in science and expanded my knowledge of future pathways for me. It has inspired me to work harder and have more patience in general. This experience will stay with me forever and continue to assist me in the future years to come. I am very grateful for the fantastic scientists at GTAC and CSL for making this experience possible.
Lexie: When I first expressed an interest in being a part of the SIRE program, I was excited. I have been to GTAC once before and my experience was incredible- I was super ready for Round 2. Doing all kinds of hands on work was a privilege, this protein and its properties are still being researched by CSL so our project could not be more relevant and applicable. The opportunity to work with a great group of students (and new friends) from all around Victoria made this project special, and hearing their stories and ambitions was so lovely.
With the support from all members of GTAC and CSL helpers, this week could not have gone better. Piquing the curiosity of youth in the science-field really paid off here and I would highly recommend this program for any students who have it come their way!
Mary: I initially chose to do this work experience week as a way to escape school and buy food in the city. My opinion on this work experience opportunity changed for the good after day 1. This engaging program has taught me way more than what school has taught me on the topic of Biology. Furthermore, getting to know others and the CSL scientists (especially Tony) has opened up my eyes to a broad range of careers related to science - nothing today ever just falls under one category; every aspect of science is fused together to continue scientific advancements in the modern world. Now I can go back to school and boast about how I was able to isolate an antibody.
Sandy: This program was one of the most interesting and immersive ones that I have been lucky to attend to for the past week. I have learned a lot about the process of conducting experiments and why we do them, in terms for medical and technological advantages. What I found the most enriching is that I am - as well as all the other students - are part of this project at CSL and if this antibody were to be developed into a medicinal drug, we would have potentially been part of this process from the very beginning. It has given much more insight in biomedicine and seeing that a lot of science disciplines link to each other in many ways.