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Gene cloning question

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Gene cloning question

Postby thewax » Sun Dec 14, 2008 6:12 am

1) How do you get the plasmid ( = how do you isolate the bacterial plasmid from cells) out of the cell? If not from cells (although Campbell and Reece Biology says there are on page 387) where do they come from? 2) Some books say that antibiotic resisting genes are already in the plasmid and some are not. (In Campbell and Reece Biology 7th edition, please refer to page 387 bullet point (1)) If yes, then they naturally have it, but if they naturally have it, then how can they tell it from those that don't have recombinant DNA (in gene cloning) in the end. If no, how do they get there? If yes, how do people engineer the gene to be placed in the plasmid?
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Postby MrMistery » Sun Dec 14, 2008 3:44 pm

Please, work through your homework by yourself. People on this website will help you, but you need to show some work first.
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Re:

Postby thewax » Sun Dec 14, 2008 7:27 pm

MrMistery wrote:Please, work through your homework by yourself. People on this website will help you, but you need to show some work first.


Please, can't you tell that this is not homework? And this is not math - how do I "show my work" on something I completely don't understand?????
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Re: Gene cloning question

Postby blcr11 » Mon Dec 15, 2008 2:46 pm

The original plasmids were naturally occuring, extra-chromosomal pieces of DNA molecular biologists started to notice right around the same time they began to appreciate the mechanism of bacterial conjugation. There is an OK Wiki article that gives a very very brief overview of plasmids: http://en.wikipedia.org/wiki/Plasmid Another decent source, though dated now, is Gunther Stent's Molecular Genetics

The old-style way to isolate plasmids took advantage of two facts: 1) DNA is more stable toward alkali (base) than RNA; and 2) plasmids are much smaller than the bacterial chromosome (although cosmids can get quite large). In very brief outline, you grew the bacterial cells containing the plasmid to high density-usually taking advantage of antibiotic selection; lysing the cells by treatment with alkali; adding solid cesium chloride and spinning the crude extract/CsCl mixture at 35-75,000 rpm for something like 12-24 hours. Under these conditions, cesium chloride forms a density gradient such that, provided you added the correct amount of CsCl, will band the plasmid in one place, and the chromosomal DNA in another place along the gradient. The RNA should have been digested by RNAse as well as by the alkaline treatment and should pellet at the bottom of the gradient, safely out of the way. When you carefully remove the bands, you get fairly pure plasmid DNA, sometimes in very high yields. Now days, you don't usually do CsCl gradients. The kits are designed to filter or pellet the larger chromosomal DNA, and the columns are much more selective for DNA, and will retain the DNA plasmid while any contaminating RNA and what not gets washes off. When the column is eluted, you get your pure plasmid DNA.

Most vectors in use today are derived from earlier, natural plasmids, though many have been so highly engineered as to be almost unrecognizable if compared side-by-side with the originals. It is common to piece together different pieces of plasmids from different sources to generate a plasmid for a specific cloning or expression purpose.
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Re: Gene cloning question

Postby thewax » Wed Dec 17, 2008 5:37 am

thanks! :D
can anyone confirm blcr11's answers?
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Re: Gene cloning question

Postby thewax » Wed Dec 17, 2008 5:43 am

Can someone also explain what blcr11 means (i.e. what RNAse is and what it does, etc.)?
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Re: Gene cloning question

Postby thewax » Sat Dec 20, 2008 11:03 pm

can someone please help?
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Postby MrMistery » Fri Dec 26, 2008 9:46 pm

you can find out what RNase is by googling it.
And really, you should try to understand the basic processes in biology before attempting to answer difficult questions.
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Postby kk » Wed Jul 29, 2009 8:07 am

Let me continue with molecular cloning.

When you generate N-fusion proteins (our "gene" of interest is fused to the N-term to the reporter gene), does it matter if you leave the Kozak sequence and a start codon in between the two coding regions? If it's OK how come that there is no reporter protein being produced alone?

Another question: how many extra amino acids should we allow to have in extra in between our gene of interest and the reporter gene - without problems. I could imagine for example if we had many (5-6-8-10-12..) it could accidentally become an NLS or other signal and our fusion protein will mislocalize.
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Re: Gene cloning question

Postby MrMistery » Sat Aug 01, 2009 3:08 am

if you leave the kozak sequence it shouldn't matter, you would just end up with a longer linker.

well it all depends: some proteins may require the N- or C- terminus to be able to move around in order to fold right. However, you're probably best off starting with 5-10. Also, most of these signal peptides are at the N- or C- terminus, not in the middle of the protein, and the probability of you creating a signal sequence out of chance is really really small.
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Postby Eous » Mon Aug 03, 2009 2:57 am

you wouldn't want to leave the Kozac sequence and start codon in between the two coding sequences just incase the genes should spit, which there is a chance of if you're trying to splice the gene into a larger sequence or if it is just a plasmid. because then you might have reporters getting made without the protein of interest (false reports) or proteins of interest that are not 'reported'. the chance of them splitting is not always significant, but if they do it could seriously mess things up for you. if there is only one kozac and start codon, then then split would nullify one gene (the one that no longer has the stuff to start with) so you would only have to worry about one of your problematic situations. i think you'd be better off with genes of interest getting transcribed without reporters cause usually you're segretating things afterwards based on the reporters (like trying to grow colonies of bacteria based on resistances and such) so if you start with the gene of interest and then the reporter, you won't have incorrect cells in your batch). at least, that's how i see it.
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Postby kk » Mon Aug 03, 2009 9:44 am

Thank you. Interesting argument on the extra-Kozak and start codon...


So is it even better to leave a few extra amino acids as for a linker in a fusion protein? First I thought the best is if the reporter sequence and "our protein" is right next to each other. It makes sense though that extra amino acids would allow correct folding.
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