pBLU and pBluescript

[Towa]

Are pBLU and pBluescript different?

[Koeng]

Yes. pBLU has the full lacZ gene, which honestly I have never seen in a plasmid. pBluescript is much smaller and therefore more efficient for cloning purposes. 

-Koeng

On Tuesday, May 10, 2016 at 9:20:35 PM UTC, Towa wrote:

Are pBLU and pBluescript different?

[Towa]

Thanks Koeng!

[Scott]

Hi Towa,

The LacZ gene can be broken in two expression units - LacZ-alpha and LacZ-omega. The pBluescript (and pUC series) plasmids, as Koeng pointed out, has the much smaller LacZ-alpha gene. LacZ-alpha by itself will not give you beta-gal activity. If you use this then you will need a bacterial host (e.g. DH5-alpha) that contains the omega gene fragment - lacZΔM15. The combination of the alpha and omega gene products produces functional beta-gal activity in what is call alpha-complementation. So it depends on what you want to do in your experiment as to which plasmid to use. Full length lacZ will allow you to express beta-gal in almost any cell type but if you want to do blue/white selection then you will need alpha-complementation with the appropriate plasmid and host.

Cheers,

Scott

http://www.opensciencenet.org

[Towa]

Hi Scott,

thanks for the explanation. I actually did some reading on the lac operon and learned about the alpha-complementation last night. It's pretty neat! 

so to summarize, pUC18 in DH5-alpha. X-gal for the lacZ to eat, and IPTG to induce the lacZ. correct? 

If I were to stick a gene with a T7 promoter in the MCS of pUC18, I won't have to add the IPTG to the agar right? 

[Scott]

Yes, pBluescript or pUC18 in DH5a with X-gal/IPTG will permit blue/white selection. If you clone (almost) anything (including a fragment with T7) into the MCS then you will inactivate the plasmid-born LacZa gene. I say almost because quite some time ago I PCR-amplified a small fragment and aimed to clone it into pUC13 in a specific direction relative to the polylinker but never got white colonies in that orientation. Turned out the fragment was one continuous ORF when inserted into the LacZa!

[Towa]

oh man, I hope I'm not as unfortunate! How does that happen though? did your fragment not include a stop codon?

[Scott]

Actually it wasn't DNA from a known coding region. It was intronic sequence that just happened to have an ORF over the length of the ~150-200bp fragment.

[Koeng]

I've also gotten various shades of blue. Usually they are lighter than the fully blue colonies so you can tell the difference.