Christos Katsifis, Week 4, Week 5
These eventful two weeks have proven to be pivotal; I have taken over yet another project. On top of the Anopheles funestus optimization, the modelling of a gene drive system, and the cloning of Homing Endonuclease Genes (HEGs), I have been directed to create a control procedure for creating variants of the AGAP007280 HEG.
In Week 3, I was assigned the cloning of a specific version of the AGAP07280 (7280 for short) gene. This was the 7280(2) version. Once I had cloned the gene through a series of transformations and bacterial plasmid extractions through mini-prep and maxi-prep techniques, I was shown many different variants and modifications of the gene. Some of the variants included a change in the HEG itself, others included MEGATAL endings, thus increasing the length of the sgRNA recognition site, and the most extreme modifications included a complete change of endonuclease enzyme within the HEG. Regardless of the change, there was no standardized procedure from creation of the 7280 HEG to the final injection of the plasmids in to the embryos of mosquitoes.
During this two week duration, I had countless Gibson reactions fail, blank gels, and non-transgenic larvae. But at the end of Week 5, I had finally solidified the control procedure for testing these versions of the 7280 HEG.
The controlled procedure basically works as a cycle: the base HEG is cloned simultaneously with the creation and cloning of the modified HEG. Along with the cloning of Week 3's versions of the 7280 HEG, I have tested the p[PR<] beta2- 7280(3) and p[PR<] beta2- 7280(3) MegaTal plasmids which will be injected within embryos at the beginning of Week 6.
There has also been much progress on the Spanish Slug gene drive model. I had received the genome and transcriptome from the DNA Database of Japan and Dr. Lubec, a researcher out of Spain who specializes in finding the function of the genes within slugs. Receiving these documents has vastly expedited the creation of the gene drive system. I had decided to go a little overboard with the system; I have selected a multiplexed Cpf1 endonuclease to target the gene responsible for the rapid metabolism and the heavy excretion of slug slime upon motion. Both of these traits are responsible for the prolific invasive ability of the slugs. I have chosen the Cpf1 endonuclease because it is easily multiplexed, and cuts at a site which is around 20-25 base pairs down from the recognition site (this drastically decreases mutative resistance to the gene drive). I have found various genes responsible for these traits, but whether they are ultraconservative is unknown to me. But besides finding the perfect gene, the sequence containing the Cpf1 gene has been established.
Side note: I would include pictures within my lab, but we are not allowed to take our phones out of our pockets due to safety restrictions. I have managed to snap a couple of photos, but I will save them for some later posts.
Outside of the lab, I have finally established a weekly routine, so the extracurricular stuff has not been too different than prior weeks.
The only difference from the regular routine was my trip to Greece from July 21st to the 25th. I was lucky enough to spend my 17th birthday (the 21st) in Greece with my cousins. My crazy cousin Jimmy again proved his craziness. I stayed in Athens for a day at the beginning and end of the trip, while traveling to the island of Ikaria for the three days in between. I had been longing for the beach this summer for I had not gone, so my two day intake of some sunburn and salt had sufficed.
In Week 3, I was assigned the cloning of a specific version of the AGAP07280 (7280 for short) gene. This was the 7280(2) version. Once I had cloned the gene through a series of transformations and bacterial plasmid extractions through mini-prep and maxi-prep techniques, I was shown many different variants and modifications of the gene. Some of the variants included a change in the HEG itself, others included MEGATAL endings, thus increasing the length of the sgRNA recognition site, and the most extreme modifications included a complete change of endonuclease enzyme within the HEG. Regardless of the change, there was no standardized procedure from creation of the 7280 HEG to the final injection of the plasmids in to the embryos of mosquitoes.
During this two week duration, I had countless Gibson reactions fail, blank gels, and non-transgenic larvae. But at the end of Week 5, I had finally solidified the control procedure for testing these versions of the 7280 HEG.
The controlled procedure basically works as a cycle: the base HEG is cloned simultaneously with the creation and cloning of the modified HEG. Along with the cloning of Week 3's versions of the 7280 HEG, I have tested the p[PR<] beta2- 7280(3) and p[PR<] beta2- 7280(3) MegaTal plasmids which will be injected within embryos at the beginning of Week 6.
There has also been much progress on the Spanish Slug gene drive model. I had received the genome and transcriptome from the DNA Database of Japan and Dr. Lubec, a researcher out of Spain who specializes in finding the function of the genes within slugs. Receiving these documents has vastly expedited the creation of the gene drive system. I had decided to go a little overboard with the system; I have selected a multiplexed Cpf1 endonuclease to target the gene responsible for the rapid metabolism and the heavy excretion of slug slime upon motion. Both of these traits are responsible for the prolific invasive ability of the slugs. I have chosen the Cpf1 endonuclease because it is easily multiplexed, and cuts at a site which is around 20-25 base pairs down from the recognition site (this drastically decreases mutative resistance to the gene drive). I have found various genes responsible for these traits, but whether they are ultraconservative is unknown to me. But besides finding the perfect gene, the sequence containing the Cpf1 gene has been established.
Side note: I would include pictures within my lab, but we are not allowed to take our phones out of our pockets due to safety restrictions. I have managed to snap a couple of photos, but I will save them for some later posts.
Outside of the lab, I have finally established a weekly routine, so the extracurricular stuff has not been too different than prior weeks.
The only difference from the regular routine was my trip to Greece from July 21st to the 25th. I was lucky enough to spend my 17th birthday (the 21st) in Greece with my cousins. My crazy cousin Jimmy again proved his craziness. I stayed in Athens for a day at the beginning and end of the trip, while traveling to the island of Ikaria for the three days in between. I had been longing for the beach this summer for I had not gone, so my two day intake of some sunburn and salt had sufficed.
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| A picture of the sunrise from Ikaria |
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| This is my crazy cousin Jimmy |
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