Erratum: 1:45 pBR322 origin is technically the MB1 plasmid origin. MB1 origin is like ColE1 plasmid origin - being an "ori" type. Details on the ColE1/MB1 replication origin can be found at ruclips.net/video/2pAM-FTFI2c/видео.html
I am assuming it would lose the dominant origin of replication so it would drop to the plasmid origin copy number of 1-2. Dumb question. I see the genes are pointing in a certain direction. That means they need to be transcribed in that direction right? Also if two genes are facing opposite directions they must be on opposite sides of the DNA correct? And lastly the DNA will still have strands running in opposite directions in a circle right? So which one is the upside down one when you lay the plasmid out perfectly like the diagrams and why does it orient like that every time?( Unless it can be either way)
Yep, it would lose the dominant pBR322 origin. But importantly, if grown with Cre, the pNS582 vector will split into two plasmids - one with pBR322 and one with P1 unit copy origin - making the entire cloning process useless. And yes, genes pointing towards certain direction means they are transcribed in that direction - two genes can be opposing to each other or facing each other, or any other combination. The orientation of the genes in a plasmid can be written in any orientation as you want. To keep it simple to follow, I fix my orientation and never change it. But I can also write the orientations as the mirror image and then the orientation would be reversed - it is still correct but for the sake of the video it can be confusing if I suddenly switch the orientation. As long as you are consistent with how you label and orient, it does not matter if you specify the forward or reverse strand. One trick to orientation is to look for reference spots. For instance, in this video, LoxP direction can serve as a reference point for the orientation of every other feature/gene in the plasmid. If I flip the orientation of the LoxP sites, then every other feature will also need to be changed so the orientations match with respect to the loxP sites. Hope this helps.
Erratum: 1:45 pBR322 origin is technically the MB1 plasmid origin. MB1 origin is like ColE1 plasmid origin - being an "ori" type. Details on the ColE1/MB1 replication origin can be found at ruclips.net/video/2pAM-FTFI2c/видео.html
When will you post lecture for expression vectors
I am assuming it would lose the dominant origin of replication so it would drop to the plasmid origin copy number of 1-2. Dumb question. I see the genes are pointing in a certain direction. That means they need to be transcribed in that direction right? Also if two genes are facing opposite directions they must be on opposite sides of the DNA correct? And lastly the DNA will still have strands running in opposite directions in a circle right? So which one is the upside down one when you lay the plasmid out perfectly like the diagrams and why does it orient like that every time?( Unless it can be either way)
Yep, it would lose the dominant pBR322 origin. But importantly, if grown with Cre, the pNS582 vector will split into two plasmids - one with pBR322 and one with P1 unit copy origin - making the entire cloning process useless.
And yes, genes pointing towards certain direction means they are transcribed in that direction - two genes can be opposing to each other or facing each other, or any other combination. The orientation of the genes in a plasmid can be written in any orientation as you want. To keep it simple to follow, I fix my orientation and never change it. But I can also write the orientations as the mirror image and then the orientation would be reversed - it is still correct but for the sake of the video it can be confusing if I suddenly switch the orientation. As long as you are consistent with how you label and orient, it does not matter if you specify the forward or reverse strand.
One trick to orientation is to look for reference spots. For instance, in this video, LoxP direction can serve as a reference point for the orientation of every other feature/gene in the plasmid. If I flip the orientation of the LoxP sites, then every other feature will also need to be changed so the orientations match with respect to the loxP sites.
Hope this helps.
♥