2 replies to this topic

[Julio-Claudian]

Dear all,

Could someone please explain how MCS are included into the lacZ gene? Or are they naturally occurring? We all understand the concept of blue-white screening where a disrupted lacZ gives only white colonies whilst intact lacZ give blue ones.

What about the MCS? Wouldn't it affect the lacZ in some way that the reading frame is altered since the insertion of different RE sites?

I tried to reason with myself that these RE sites are naturally occurring but I happen to see a few examples of pGEM plasmids (pGEM-7Zf, -11Zf and -13Zf) having different MCS in the lacZ gene.

Reason I ask is that I wanted to include a few RE into an existing gene but not wanting it to lose its function.

Thanks people.

Edited by dreamchaser_jc, 26 April 2010 - 09:26 PM.

[phage434]

The MCS of these plasmids are highly engineered, by clever insertion of restriction sites.  This was done in such a way that the coding sequence for the protein coding region is either preserved, or mutated with changes not affecting function.  It is remarkable that this was done largely at a time prior to PCR as a useful technique for manipulating DNA.  You can do this too, and will have a much easier time.  Several programs, such as the gene design program at DNA 2.0, allow you to insert and remove restriction sites from protein coding regions relatively easily, but not with the overlapping complexity of the normal MCS regions.

[Julio-Claudian]

Thanks phage434

Will have a look at the program. Am looking at the possibility of inserting 4 new RE sites not present in the aminoglycoside 3'-phosphotransferase (neo) gene; or any other genes in the future..

Edited by dreamchaser_jc, 28 April 2010 - 07:04 PM.