A Scrapped Cloning Story
ANONYMOUS
Photo by Warren Umoh on Unsplash
I joined S’s lab to pursue my PhD. The lab works on the effects of amyloids on protein homeostasis among other things. The lab has been solely dependent on mammalian cell culture as a source of substrate to do the experiments. But, because of my previous experience with mammals, S pushed me to work with mouse models.
I was assigned the task to clone the DNA of a model amyloid, α-synuclein, and three of its mutants associated with Parkinson’s disease in a plasmid which are compatible to express specifically in the dopaminergic neurons in the brain.
For those uninitiated in molecular biology techniques, cloning some DNA fragment is a different ballgame from cloning an entire organism. Cloning DNA just means creating multiple copies of it to study its products further.
So, back to the story: we purchased the vector DNA plasmid. The DNA that would code for α-synuclein and its mutants was already available in the lab in other plasmids. With the confidence of an amateur, I started the cloning of all four variants at once.
3D structure of alpha-synuclein
Image credits to Ganesh Mohan T, CC BY-SA 4.0, via Wikimedia Commons
One of my seniors gave me a suggestion as I already have the variants cloned, why don’t I just convert them to the original by changing a base pair of the DNA fragment. It was a brilliant idea. There was only one problem. I already had ordered the primers twice for this cloning.
Making S order yet another set of primers for the same cloning was a tough task, tougher than the cloning itself."
"
I separated the DNA of the α-synuclein variants from the available plasmids and joined it with the new plasmid. Lo and behold! I was successful… but well, not completely. I was able to clone one of the variants on the first go. Contrary to S’s opinion, that was motivating for me. After a few more tries, I was able to clone two other mutants, but the original α-synuclein was not getting cloned. I remade the working dilution of primers, reordered the primers, redesigned the primers with different restriction enzyme sequence, but the original one was not getting cloned.
I prepared a powerpoint presentation with the agarose gel images and Sanger sequencing results of all the failed attempts and sent it to him. I took a deep breath and went to his cabin. I presented my failed results on his computer. After 10 min of ridicule, he agreed to order the new set of primers, but only after taking a false promise to see the positive result within a week of arrival of the primers. I happily exited the cabin.
Cloning the variants/ mutants: a success story
Image prepared by Aswathy in consultation with Anonymous
Cloning alpha-synuclein: a disaster
Image prepared by Aswathy in consultation with Anonymous
These ordered primers were not the usual 20-25 base pair primers to clone a sequence. These were 40+ base pairs long with a mismatch to the template near the centre. I was going to synthesise the rest of the gene with the plasmid. The whole synthesis was approx. 7200 base pairs. For this heavy work for each molecule, I needed high fidelity expensive enzymes to synthesise the DNA, which I had already ordered under the nose of S without him noticing. I did the 6 hrs long synthesis reaction and ran the agarose gel. I was able to see a smear. I was disappointed. I showed it to my senior and they told me that it does not look perfect, but it could work. I excised the piece of agarose gel where the synthesised product was expected and proceeded to amplify it in bacteria by transformation. I did get a few bacterial colonies which, on testing, turned out to be negative for the desired gene. I repeated this synthesis more than five times. One time, I even got a positive band which on testing turned out to be the mutant from where I started.
After months of trying, S gave up on the hopes of my ability to clone the gene. It was 7 years ago. The gene has still not been cloned to this day. That project has been scrapped.
Editorial Note
This piece by Anonymous is an ode to the thousands of failed experiments/projects that never see the light of the day, and never get talked about.
About Anonymous
Anonymous completed their PhD from a premiere research institution in India.
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