Re: [DIYbio] Re: Clustered essential genes, any ideas?

@ Mega I don't have enough money to synthesize a minimal genome :( that's for Craig Venter to do. I am using a positive and negative selection cassette. I'll insert it into regions without essential genes then use long primers (60-100bp) to remove the cassette by homologously recombining near 2 essential genes, deleting the entire region around them (I have planned 52 of these, to delete more than 3mbs). Yea some genes are gonna get expressed more/less, but in the "Combining 2 genome" papers that didn't seem like a huge problem. And it probably is an island from its ancestors, but perhaps adapted for something else...

@ Lisa I think that was just what I was looking for :D Thank you a ton! All of the evidence is in support of that theory. It turns out DNA replication proteins (hence those replication proteins are increasing their own transcription), ribosome proteins, and RNAs is what is mainly there. It looks like most the tRNAs and rRNAs are there actually, then there is fewer and fewer as it gets closer to the terminus. There is still one near the terminus, but it looks like the majority of RNAs are there near the origin, or halfway between the origin and the terminus. Meanwhile the ftsZ ring is 400kb from the terminus, which is pretty nearby in the big scheme of things. I read a paper a while ago (I will try and dig it up) that explained how the ftsZ ring's transcription stayed stable and that when times were good that the chromosomes began replicating more and more and blocked ftsZ ring formation for splitting, and this was an adaptation for quick growth. Since there is some "marker genes" I could use for this analysis (ftsZ, dnaA, ect) I think I will begin looking through slow and fast growing bacterial genomes to find if there is any correlation. Thanks a ton!!!

On Sunday, March 23, 2014 2:21:49 AM UTC-7, Mega [Andreas Stuermer] wrote:

Maybe this island is the remnant of its ancestors. That then duplicated and mutated. 

If you have the minimal genome synthesized at one time, remember that you'll need a marker for the cells. I recommend lux :D 

So the minimal genome bacillus would glow by default (already very cool! but, it's getting even better - wait for it). When you do homologous integration, by knocking in your gene of interest you would knock out luxAB. So you can see that your experiment worked.  :D 

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