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Choosing vector plasmids and subcloning 

the bumbling biochemist
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When you order a cDNA plasmid (the DNA copy of the mature messenger RNA (mRNA) instructions for making a protein) from a repository (e.g. addgene, DNASU), it will likely come in some sort of generic cloning plasmid. And you will want to “subclone” it into an expression plasmid if you want to use it to make protein from it (i.e. move it out of the one plasmid and into another). blog form: bit.ly/vector_...
There can be a lot of options and weird nomenclature and stuff when it comes to plasmids, but the key things they differ in are usually:
- copy number - how many copies of the plasmid will each cell host (high’s good for cloning, lower for expression)
- promoter usage - how will you tell the cells to use an RNA Polymerase to make mRNA copies of the gene (and subsequently protein from those mRNA instructions) (T7 (w or w/o lac), tac, T5)
- inducible expression of the RNA Pol? (e.g. induce T7 expression with IPTG)
- selection markers (typically antibiotic resistance genes for Amp, Kan, Strep, etc.)
- potentially secretion signals
- epitope tags (His, Flag, etc., which can be at the N-terminus (start of the protein) or C-terminus (end of the protein))
- often with protease cleavage sites (TEV, HRV3C, thrombin, etc.) for removal from protein
- restriction enzyme cut sites for cloning with restriction enzymes (often there are multiple of them in a region called a multiple cloning site (MCS))
- the different letters after a plasmid number (e.g. pET28a vs pET28b vs. pET28c) usually refer to what reading frame the inserted protein will be read in with respect to the cut site
In terms of reading frames, which plasmid you start with is less of an issue if you’re using a PCR-based cloning strategy (such as SLIC). PCR-based strategies are also good because they let you do “scarless” cloning - you don’t have extra letters on the ends of your proteins that come from having some of the MCS still there. PCR-based strategies are also great because you can easily clone in different tags and things, and do things like swap the tag from the N- to the C-terminus or vise versa, which can sometimes make a difference in terms of tag accessibility, protein folding, etc.. More on cloning methods here: bit.ly/molecula...
plasmids will often have a bunch of notation with things like Δsomething (Δ is delta and it means something is missing, missing things might also be indicated with a superscripted - sign)
- for example, recA- cells are deficient in an E. coli repair system - this system carries out homologous recombination, which can shuffle things around, which you might not want
- an endA mutation makes cells endonuclease I deficient - they don’t make a nonspecific endonuclease (nucleic acid cutter) that’s E. coli normally keep in their periplasmic space - this mutation can keep your plasmid from getting degraded
another thing to look for is the potential for blue-white screening: bit.ly/bluewhi...
some bacterial plasmids are designed for blue-white screening, including: pGEM-T, pUC18 and pUC19, & pBluescript 
but the host cells need to be compatible with it too - some that are: XL1-Blue, DH5α, DH10B, JM109, STBL4, JM110, & Top10 
When looking for a cDNA clone, the first place I normally look is Addgene. Although it sounds like it would be some commercial entity just out there to make a profit, it actually serves as a nonprofit plasmid repository - labs can send a sample of their plasmids to and Addgene will propagate them (make more copies) and distribute them to the public for a minimal fee ($75/plasmid when I just ordered some).
Addgene is a great place to start because, if authors have deposited the plasmids they used for protein expression, and that expression was in the system you want (e.g bacterial expression not a mammalian expression vector) then you won’t even have to subcclone! and the plasmid might be optimized for good expression - or at least you know it should work). If you can only find the gene cloned into a vector for a different expression system, don’t worry - you can just subcclone it into one you want - more work, but shouldn’t be an issue - and definitely not worth paying commercial companies an arm and a leg to get a version in the plasmid you want.
note: Addgene also has a really great educational blog - I suggest checking out their Plasmids 101 guide www.addgene.or...
Another source which you can turn to if Addgene turns up short is DNASU, which is a depository that has plasmids containing cDNAs for “all” genes, even those that people haven’t worked with already. see comments for rest

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17 авг 2024

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Комментарии : 6   
@Sid00077
@Sid00077 2 года назад
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
@thebumblingbiochemist 2 года назад
Thanks! Sorry about that!
@Sid00077
@Sid00077 2 года назад
@@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!
@thebumblingbiochemist
@thebumblingbiochemist 2 года назад
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
@hungsangchan
@hungsangchan Год назад
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.
@thebumblingbiochemist
@thebumblingbiochemist Год назад
Thanks! Glad you found it helpful. Cool project! I recommend checking out addgene's cloning blog. Best of luck!
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