I am not sure I understand you. We will be transferring a gene from a wild type rhodococcus to a plasmid, and the plasmid into e. coli. Kind of like a biobrick part, minus the standard ends. LacZ' fragments are also genes of interest for use in constructing plasmids- it has a pretty easy functionality to test for, so it might be fun to yank out of e. coli and stick into a plasmid. Or a restriction enzyme producing plasmid- that could be fun.
You are completely correct about the pigment vs. lacZ+X-Gal argument though. It would make a sweet cloning vector.
There is no need to score a "pigment + phenotype" because we are copying genes out of rhodococcus into e. coli, and the rhodo should have the gene (it has been sequenced).
--A
On Mon, Jun 4, 2012 at 4:44 AM, Srdjan Gavrilovic <sasvimlogicno@gmail.com> wrote:
Hi Avery,
Im sorry but I cant join you organizing the class but I have few suggestions to make.
"a pigment gene from a Rhodococcus"..."I have done this before, but not at bosslab."
Im not sure how easy it is to distinguish transgenic bacteria from wild type. If scoring is easy I would say that it would be a great idea to try and engineer the gere for future cloning eforts.
The thing is that for cloning into LacZ, people have to use blue-white screening. To score a potentially right clone use of IPTG and X-gal is needed. Getting and dissolving it is a real pain.
Adding multi cloning site into a pigment gene and redesigning any of cloning plasmids (exchanging LacZ with MCS for a pigment gene with MCS) would be a great brake through for DIYbio. People could start doing cloning easier.
Restrict plasmid, attempt to ligate an insert, transform into e.g. E.coli and take colonies that do not produce pigment (if pigment is present it means that ORF is restored and there is no insert), test if they have the right insert and do what ever they want to do with their gene of interest.
I do think that its nice to educate people ubut showing them how to clone plus building a great tool for the DIYbio people at the same time. I would say that you might get a lot of people wanting to help and to have such a plasmid for private cloning purpose. Taking a course and going home with a plasmid like that...Even academic labs would want to have such plasmid.
Now the thing is. How easy it is to score a pigment positive phenotype in E.coli?
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