Hey there, this is a great explanation and I really appreciate you putting it out on the internet. My only criticism would be that, since there is so much information on the screen, it can be a bit hard to follow. Therefore, my only advise will be to put them in some kind of order (maybe number the sub-headings). Apart from that, you did a great job at explaining things. Thank you!
@@thebumblingbiochemist no need to apologize at all. As someone who worked with Drosophila for the last 3 years and is now transitioning to cell culture and bacteria, your content is very helpful!
These are generally in generic cloning vectors but you can easily subcclone them out. Note: you might see different splice versions or alternative transcripts for the same gene, so you might need to do a little looking in UniProt, etc. to make sure you get the version you want. Note 2: some plasmids are marked “fusion” and others “closed” - “fusion” ones don’t have stop codons - these must be supplied by the vector you’re cloning into, “closed” ones do have stop codons I’ve mainly used pET vectors, chiefly pET28 vectors. I’ve always been curious whether these were really optimal for expression or just what people started using (in the 1980s starting with Studier et al.) and then people used them because others used them and companies (Novagen & Invitrogen) starting making lots of variants of them. So people started buying them and… When I was looking into the matter, I came across this paper from Patrick Shilling et. al describing an optimized pET vector - apparently the original one, though great, had some design flaws including a truncated T7 promoter (compared to the “consensus” one that’s naturally used) and a suboptimal translation initiation region (TIR). They restored the T7 promoter to its full length & tested out a bunch of TIR sequences and found a version that was best for expression of the reporter gene they were using in their test (superfolder GFP). And they deposited this plasmid (pET28a T7pCONS TIR-2 sfGFP) in Addgene if anyone wants to test it out (you can also try cloning the changes into other pET vectors): www.addgene.org/154464/ And here’s that paper: Shilling, P.J., Mirzadeh, K., Cumming, A.J. et al. Improved designs for pET expression plasmids increase protein production yield in Escherichia coli. Commun Biol 3, 214 (2020). doi.org/10.1038/s42003-020-0939-8 As well as the original Studier et al. paper: Rosenberg, A. H., Lade, B. N., Chui, D. S., Lin, S. W., Dunn, J. J., & Studier, F. W. (1987). Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene, 56(1), 125-135. doi.org/10.1016/0378-1119(87)90165-x GenScript vector list - a good table of common vectors used for mammalian, bacterial, insect, & yeast expression: www.genscript.com/expression-vector-selection-guide.html for more information: Plasmids 101: Origin of Replication, Kendall Morgan, 2020 blog.addgene.org/plasmid-101-origin-of-replication Plasmids 101: Stringent Regulation of Replication, Jason Niehaus, 2015 blog.addgene.org/plasmids-101-stringent-regulation-of-replication GenScript vector list: www.genscript.com/expression-vector-selection-guide.html Addgene: www.addgene.org/ DNASU: dnasu.org/DNASU/Home.do
Hi there - great video! thank you for such an informative and accessible explanation of plasmids vectors. I'm actually just starting out in the UK as a PhD student looking at developing an e.coli- and b.subtilis-based fluorescence reporter gene biosensors. How do I know which strain of, let's say, e.coli is compatible with a particular plasmid of interest? How do I know whether a low copy plasmid will be compatible with the genomic machinery necessary for a particular strain of bacteria to produce copies of that plasmid? Aside from T7 systems, how else might a particular strain of e.coli be incompatible with a particular plasmid, or vice versa? Thanks in advance.
Hey there, this is a great explanation and I really appreciate you putting it out on the internet. My only criticism would be that, since there is so much information on the screen, it can be a bit hard to follow. Therefore, my only advise will be to put them in some kind of order (maybe number the sub-headings). Apart from that, you did a great job at explaining things. Thank you!
Thanks! Sorry about that!
@@thebumblingbiochemist no need to apologize at all. As someone who worked with Drosophila for the last 3 years and is now transitioning to cell culture and bacteria, your content is very helpful!
These are generally in generic cloning vectors but you can easily subcclone them out. Note: you might see different splice versions or alternative transcripts for the same gene, so you might need to do a little looking in UniProt, etc. to make sure you get the version you want. Note 2: some plasmids are marked “fusion” and others “closed” - “fusion” ones don’t have stop codons - these must be supplied by the vector you’re cloning into, “closed” ones do have stop codons
I’ve mainly used pET vectors, chiefly pET28 vectors. I’ve always been curious whether these were really optimal for expression or just what people started using (in the 1980s starting with Studier et al.) and then people used them because others used them and companies (Novagen & Invitrogen) starting making lots of variants of them. So people started buying them and… When I was looking into the matter, I came across this paper from Patrick Shilling et. al describing an optimized pET vector - apparently the original one, though great, had some design flaws including a truncated T7 promoter (compared to the “consensus” one that’s naturally used) and a suboptimal translation initiation region (TIR). They restored the T7 promoter to its full length & tested out a bunch of TIR sequences and found a version that was best for expression of the reporter gene they were using in their test (superfolder GFP). And they deposited this plasmid (pET28a T7pCONS TIR-2 sfGFP) in Addgene if anyone wants to test it out (you can also try cloning the changes into other pET vectors): www.addgene.org/154464/
And here’s that paper: Shilling, P.J., Mirzadeh, K., Cumming, A.J. et al. Improved designs for pET expression plasmids increase protein production yield in Escherichia coli. Commun Biol 3, 214 (2020). doi.org/10.1038/s42003-020-0939-8
As well as the original Studier et al. paper: Rosenberg, A. H., Lade, B. N., Chui, D. S., Lin, S. W., Dunn, J. J., & Studier, F. W. (1987). Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene, 56(1), 125-135. doi.org/10.1016/0378-1119(87)90165-x
GenScript vector list - a good table of common vectors used for mammalian, bacterial, insect, & yeast expression: www.genscript.com/expression-vector-selection-guide.html
for more information:
Plasmids 101: Origin of Replication, Kendall Morgan, 2020 blog.addgene.org/plasmid-101-origin-of-replication
Plasmids 101: Stringent Regulation of Replication, Jason Niehaus, 2015 blog.addgene.org/plasmids-101-stringent-regulation-of-replication
GenScript vector list: www.genscript.com/expression-vector-selection-guide.html
Addgene: www.addgene.org/
DNASU: dnasu.org/DNASU/Home.do
Hi there - great video! thank you for such an informative and accessible explanation of plasmids vectors. I'm actually just starting out in the UK as a PhD student looking at developing an e.coli- and b.subtilis-based fluorescence reporter gene biosensors.
How do I know which strain of, let's say, e.coli is compatible with a particular plasmid of interest?
How do I know whether a low copy plasmid will be compatible with the genomic machinery necessary for a particular strain of bacteria to produce copies of that plasmid?
Aside from T7 systems, how else might a particular strain of e.coli be incompatible with a particular plasmid, or vice versa?
Thanks in advance.
Thanks! Glad you found it helpful. Cool project! I recommend checking out addgene's cloning blog. Best of luck!