In this video, BIOME lab manager Colin Kalicki gives a tutorial on how to use the online platform Benchling to satisfy your biology needs, including plasmid design and modeling.
Also you're a great teacher and are clearly very knowledgable and enthusiastic. You kind of remind me of Jacob Collier when he speaks about music - check him out if you don't know him, he's very gifted.
Good video, however I think you may have made a mistake with your Kodak sequence, the Methionine or ATG is supposed to fall inside of your kozak sequence, your GFP gene that you implemented included the ATG start codon for you, but you forgot to erase that and center the gfp ATG start codon with the one in the kozak sequence, and since the kozak sequence you used included an extra Guanine after the ATG, you ended up shifting your entire gene by 1 base pair, effectively breaking the gene. All you have to do to fix this however is go back in and delete the ATG of the GFP gene and the extra guanine in the kozak sequence. I would also recommend finding another yeast Kozak sequence as the one you used requires the guanine after the ATG however the GFP gene you used has an amino acid that can’t start with a guanine, so the easiest way to fix this is to delete your current kozak sequence and ATG, find another yeast kozak sequence that doesn’t have a base pair requirement after the ATG, for example ggatccacgattaaaagaATG (this is an actual yeast kozak that you can use) then after the ATG comes all of the other base pairs for the amino acids needed for the GFP protein. Hope this helps, sorry I’m not the best at explaining things, but I hope you understand what I’m trying to say. Edit: grammar
Hi, I'm not sure if anyone's gonna be able to see this after two years, but why do you have all of those dead spaces with no annotations? I just see them everywhere on the plasmids I search for and I wonder if there's usually some purpose to all those dead spaces other than "missing annotations".
Hi - we did see it and great question! Having all of the components of the plasmid too close to each other may impact the binding of proteins to those sites so you want some space for easy access! So while the unannotated spaces do not transcribe to any RNA they are still useful and needed in a plasmid backbone!
I don't know what the odds are you will read this given the time difference, but I have a question that even my deepest of internet dives can't seem to answer. What if you don't want to use a pre-made backbone,? As in how are backbones originally made/designed? Is it just taking a blank plasmid and adding in promoters and resistance genes and the like as if they were lego bricks (in which case where does the blank space and unannotated resistance enzymes come from), it is a matter of extracting the very base from an actual yeast cell and sequencing and editing, or something else? Given the analogy of computer programming libraries, I'm curious to know what's actually happening in a library as I am using it. If you read this I was hoping you could point me in the right direction.
