Christos Katsifis, Week 3
While, of course, Mr. Sham's visit was the highlight of the third week here in London, the third week of EXP has been the most productive and formative so far.
The science portion of the third week had been diverse, and by that, I mean that I have had an immense amount of more work (which is great!). Besides my work in the insectory, which pertains to finding the optimal conditions of the Anopheles funestus strain described in the last blog, I have been assigned two more projects. Although the initial regulation for me in the lab required constant and strict supervision, the rules have been disbanded due to the work I have submitted already (the gene drive paper and the preliminary results of the Anopheles funestus project).
The project I have been assigned with in the lab is involved for a majority with cloning CRISPR/Cas9 sequences within a bacteria plasmid. But to begin this project, I first had to edit the sequence slightly of the desired gene. The gene, AGAP007280, encodes for mosquito female fertility, specifically the development of the egg within females. The lab had cut that gene, at the specific target site with preset CRISPR sgRNA's, and left a blunt end for the insertion of coding for a CRISPR/Cas9 complex, while placing the gene on a plasmid, also containing multiple fluorescent markers and clindamycin resistance. This left for the insertion of a CRISPR/Cas9 complex. This gene was now called AGAP007280(2), for verison 2.
After the gene had been set, there were some malfunctions with the function of the CRISPR/Cas9 produced from the coding sequence. Using the "Program-with-no-name", or PON as they call it, I was able to compare a functional CRISPR/Cas9 sequence with the faulty one, identifying six nucleotides that were relatively abstract. I presented this to the project supervisor, Dr. Carla S... (really long and Italian), and she approved the proposed modifications. I had proposed two altered forms of this 7280 gene, a deletion of the six nucleotides, as it would be much easier and result in minimal to no difference in optimal complex efficiency, and an insertion of six nucleotides matching the compared sequence where the faulty ones were, which is self-evident. I had proceeded with a double stranded, blunt end cut of the 7280(2) gene at the front, and end of the problematic site. Next, I had done a Maxiprep kit to extract the larger segments of DNA rather than the smaller portions. After I had my selected gene, I handed the samples to Dr. Hammond, who inserted the specific mutation for the insertion sample. I then did a ligation to complete the creation of the altered plasmids. Once these plasmids were created, I had transformed them in to a sample of Top10 bacteria, then creating cultures with plates containing clindamysin. Thankfully, the bacteria grew very much over the sixteen hour period. Next, I had to do a miniprep, which extracts bacteria plasmids rather than the whole portion of genomic DNA.
All that was left was extracting the plasmid DNA, rather then the genomic DNA, but unfortunately for me, there were complications. All of the kits I had used previously had ethanol already added to the buffers; but in this one, there was one buffer which did not have the ethanol added. The only problem with that statement was that I did not know that ethanol was added to buffers prior to their use. So I used that very buffer, and lost all of the DNA, leaving me with 10 nano-grams per micro-liter, rather than 500 nano-grams per micro-liter. Everyone laughed at me. This mistake was made on a Friday, so I had no time to re-transform, re-culture, and re-extract the plasmids.
The second project I have been assigned is the computer modelling of a gene drive. I have the freedom to choose what organism, choose the purpose, and even choose the region. I ultimately would up with the Spanish Slug (Arion vulgaris), a highly reproductive and insecticide resistant three inch, bright red slug. I have yet to figure out how to create this gene drive system in that slug.
Besides the science aspect of my experience, I had been very busy outside of the lab. I have been reading a lot, I have finished about three quarters of all of the summer reading books on the list, and I have already completed all of the AP Chemistry summer assignments.
I also routinely went to the Iranian wrestling gym, luckily wrestling a 30 year old, two hundred pound Olympic wrestler in his prime. His name is Amir.
My PI played soccer with everyone from the lab too this week. Usually, the people from specifically my lab who play on Mondays are the technicians Chris and Dario, and then me. As we arrived at the soccer fields, Dario began to swear in Italian. He forgot all of his clothes at the lab. Luckily, because Dr. Nolan (the PI) had worn white compression shorts under his regular shorts, Dario was able to change from his jeans and in to the regular shorts. But unluckily, Dr. Nolan was left in his white compression shorts sparing no one. It was very funny.
The science portion of the third week had been diverse, and by that, I mean that I have had an immense amount of more work (which is great!). Besides my work in the insectory, which pertains to finding the optimal conditions of the Anopheles funestus strain described in the last blog, I have been assigned two more projects. Although the initial regulation for me in the lab required constant and strict supervision, the rules have been disbanded due to the work I have submitted already (the gene drive paper and the preliminary results of the Anopheles funestus project).
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| The mosquitoes would only bite my feet, and I was tired of it |
After the gene had been set, there were some malfunctions with the function of the CRISPR/Cas9 produced from the coding sequence. Using the "Program-with-no-name", or PON as they call it, I was able to compare a functional CRISPR/Cas9 sequence with the faulty one, identifying six nucleotides that were relatively abstract. I presented this to the project supervisor, Dr. Carla S... (really long and Italian), and she approved the proposed modifications. I had proposed two altered forms of this 7280 gene, a deletion of the six nucleotides, as it would be much easier and result in minimal to no difference in optimal complex efficiency, and an insertion of six nucleotides matching the compared sequence where the faulty ones were, which is self-evident. I had proceeded with a double stranded, blunt end cut of the 7280(2) gene at the front, and end of the problematic site. Next, I had done a Maxiprep kit to extract the larger segments of DNA rather than the smaller portions. After I had my selected gene, I handed the samples to Dr. Hammond, who inserted the specific mutation for the insertion sample. I then did a ligation to complete the creation of the altered plasmids. Once these plasmids were created, I had transformed them in to a sample of Top10 bacteria, then creating cultures with plates containing clindamysin. Thankfully, the bacteria grew very much over the sixteen hour period. Next, I had to do a miniprep, which extracts bacteria plasmids rather than the whole portion of genomic DNA.
All that was left was extracting the plasmid DNA, rather then the genomic DNA, but unfortunately for me, there were complications. All of the kits I had used previously had ethanol already added to the buffers; but in this one, there was one buffer which did not have the ethanol added. The only problem with that statement was that I did not know that ethanol was added to buffers prior to their use. So I used that very buffer, and lost all of the DNA, leaving me with 10 nano-grams per micro-liter, rather than 500 nano-grams per micro-liter. Everyone laughed at me. This mistake was made on a Friday, so I had no time to re-transform, re-culture, and re-extract the plasmids.
The second project I have been assigned is the computer modelling of a gene drive. I have the freedom to choose what organism, choose the purpose, and even choose the region. I ultimately would up with the Spanish Slug (Arion vulgaris), a highly reproductive and insecticide resistant three inch, bright red slug. I have yet to figure out how to create this gene drive system in that slug.
Besides the science aspect of my experience, I had been very busy outside of the lab. I have been reading a lot, I have finished about three quarters of all of the summer reading books on the list, and I have already completed all of the AP Chemistry summer assignments.
I also routinely went to the Iranian wrestling gym, luckily wrestling a 30 year old, two hundred pound Olympic wrestler in his prime. His name is Amir.
My PI played soccer with everyone from the lab too this week. Usually, the people from specifically my lab who play on Mondays are the technicians Chris and Dario, and then me. As we arrived at the soccer fields, Dario began to swear in Italian. He forgot all of his clothes at the lab. Luckily, because Dr. Nolan (the PI) had worn white compression shorts under his regular shorts, Dario was able to change from his jeans and in to the regular shorts. But unluckily, Dr. Nolan was left in his white compression shorts sparing no one. It was very funny.
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