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Textbook Canon: Expressing Any Genes in Bacteria


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#16 Julio-Claudian

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Posted 12 August 2012 - 08:59 PM

For the question related to is ORF=gene, I will give you a riddle (actually is the fact that made me also think about this a few years back): C. elegans has ~20000 different proteins but ~36000 genes. Where does the difference come from?

Hint: promoter/terminator are needed for transcription; rbs, start/stop codons are needed for translation. Think about the product of each process.


Love riddles and I'll take a stab at this (scoffers, hold your peace Posted Image):
  • This could have everything to do with the nature of eukaryotic genes where the RNA transcripts undergo a splicing step before translation. So presence of non-coding gene/DNA would be sort of "filtered" in the splicing step before those encoding a protein would finally be translated.
  • Using the hint, anything that comes under the control (within the confines) of the promoter/terminator pair will be transcribed. But the rbs might not be present at all. Taking this a step further, the absence of the rbs and start and stop codons could be due to a 'frame-shifted translation' (ok, I have no idea what that's called. And we arrived at the two-promoter situation).

Getting a little help, the difference in the number of genes and proteins are caused by presence of "transposons, pseudogenes, and other artifacts".

This article in EMBO reports states that

"Non-protein-coding RNA transcription in the eukaryotes falls into two classes: introns and other non-protein-coding RNAs. In humans, introns account for ~95% of the pre-mRNA transcripts of protein coding genes, and are generally of high sequence complexity"


and does seem to help my explanation in (2) that everything gets transcribed but not everything gets translated. I suspect, whilst these are facts, you're looking for something simpler with regards to promoter, terminator, rbs, and start/stop codons with the processes involved.

#17 ascacioc

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Posted 12 August 2012 - 11:20 PM

Yes I am looking for something simpler. Occam's razor: the right explanation is the simplest onePosted Image Let's stay with prokaryotes. They are simpler:) You have your answer in the article from EMBO reports. Hint: not introns.

Other hints: which types of RNA you know? which type of RNA is translated to proteins?

Andreea

#18 ascacioc

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Posted 12 August 2012 - 11:25 PM

And to not wonder off bacteria, I will revise my sentence for E. coli K-12: 4,377 genes, 4,290 proteins.:)

#19 Julio-Claudian

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Posted 13 August 2012 - 02:09 AM

Interesting.

*looks left and right* I think I'll have another go at it.

If all DNA/genes were destined for proteins, then there wouldn't be any tRNAs and rRNAs left. Or RNA I & II, and miRNAs for regulation [in C. elegans].

That should account for the difference in the number of genes and proteins.

I sure hope this is the 'simpler' answer Posted Image

Edited by Julio-Claudian, 13 August 2012 - 02:10 AM.


#20 ascacioc

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Posted 13 August 2012 - 02:17 AM

Yup:) You got it right. Not all genes make mRNA --> proteins, some genes make tRNAs, rRNAs, RNAis, piwiRNAs etc. Actually, C. elegans is the organism in which RNAis and piwiRNAs were discovered.

Why I did not accept the introns answer: if you have a gene with introns, it leads to a spliced mRNA (or several) and to a protein (or several spliced variants). In the simple terms: 5-90% of a gene gets to a protein; however when we count this gene to the toal amount of genes we do not say 0.5 gene + rest; we still say this gene coded for a protein. In the EMBO reports they state in other words that 5% of the DNA in genes leads to proteins, not 5% of number of genes lead to proteins. You know what I mean?

Andreea

#21 Julio-Claudian

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Posted 13 August 2012 - 03:55 AM

Yes I do :) thank you Andreea!

#22 pito

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Posted 14 August 2012 - 07:23 AM

Cloning vector vs expression vector: yes you are right; the difference is in having the promoter, rbs, terminator. I never used a cloning vector. The synthetic gene companies send the genes under the form of gene inserted in a cloning vector. Also some people working in generating DNA libraries are using cloning vectors because it is easier to handle (they are smaller, easier to transform, do not express the proteins from the genes, which make the bacteria happier).
With the origin of replication: I do not know why for expression you use the low copy number plasmids. I have just noticed that all my expression vectors have the low copy number plasmids. The reason that was given to me is that having too many copies per cell would lead to overproduction of the protein which is toxic to the cell. However, having strong promoters such as T7 has the same result. But maybe it is better not to change to many variables at once. For example, when I test which plasmid is better for my protein, I check different promoter strengths (among other things). If I would have different stregths of replication origins, I would change 2 variables in between experiments at once and I wouldn't know what is the cause for the difference.


Not following the entire discussion, but the discussion about how and low copy all depends on your needs.

High copy can indeed cause the formation of inclusion bodies in your bacteria, which could be bad, however it can also be good, because its easier to seperate inclusion bodies from the native proteins.
High copy is also not wanted if you are expressing potentially toxic proteins
High copy can also cause "stress" on your cells: all the energy goes to those plasmid/genes/proteins, this is not always what you want.
Low copy also has a more controled mechanism in many cases so that the progeny of your cells keep the plasmid.
Depending on the gene you are expressing, you might need other genes/proteins from the cell itself... a certain pathway , and you can create a bottleneck if enzym2 (you installed it, in the plasmid) is present at high concentration due to the high copy nature of your plasmid while enzym1 and 3 (from the bacteria itself) is present in lower amounts or works slower..
Also: you need to think about the energy balance of the organism: its ok to insert a certain enzym, but if this enzym used NADH you are recruiting NADH from other processes in your cell.. so you need to concider this too.
Same goes for ATP, NADP etc...
You cell has everything balanced.. as soon as you start influencing this......

I also wonder: what do you call low copy? Just 1 or?

An example of a study about this low copy vs high copy: http://www.ncbi.nlm....pubmed/11120644

It all depends on your needs and most of the knowledge about these things is not found in (fundamental) research, but rather in the industry (which is often not published)

Edited by pito, 14 August 2012 - 07:27 AM.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.





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