Re: [DIYbio] Translating bacterial DNA into plant DNA

I'll be doing it the coward methode.
Promotor-RBS-LuxA-Terminator -- Promotor-RBS-LuxB-Terminator ..... C-D-E

The most expensive it would be that it doesn't work. And one'd have to order it again...



In the database I found all the lux genes. The gene sequnece does not contain promoter nor RBS or terminator if it's just called "LuxA from Vibrio Fischeri strainxxxx"???? Because it does say only "gene" but not "promoter" . So I'm free to just take that seqeunce and attach a good promoter?






 

2012/6/11 Cathal Garvey <cathalgarvey@gmail.com>

Actually, generally Eukaryotes need one-promoter-per-CDS (CDS = "Coding
Sequence). So for each protein coded by the operon, you'll need to
extract the coding sequence, and provide it with its own
promoter/rbs/cds/terminator setup.

Yes, this is unfortunately very costly! It's due to the difference in
how bacteria and eukaryotes start reading mRNA: prokaryotic ribosomes
*assemble* on the RBS, whereas eukaryotic ribosomes first bind the
modified-G at the 5' end of the mRNA, and then travel downstream until
they find an RBS, whereupon they start translating. But, when they hit a
stop codon, they dissociate, so additional CDS's get ignored.

There are so-called "Internal Ribosomal Entry Sites" for Eukaryotes,
usually from viruses. I don't know if there are any for plants, or for
your target species of plant. They are often virus-derived, and may act
as a warning signal to plant innate defence systems. I simply don't
know. If you do find any "IRES" for your plant species in the
literature, it would save you a lot of extra DNA, because you could then
re-design the operon to work as an operon in plants, instead of needing
~8 distinct genes!

Consider however: A lot of the genes in the Lux operon code for proteins
that often work in tight association together. For example, LuxAB form
the actual luciferase, which does the job of digesting mature luciferin
to create light. LuxCDE work to create mature luficerin. I think that
LuxFGH are regulatory genes, LuxI certainly is: You mightn't need these
if you replace them with a plant regulatory signal instead.

If that leaves only LuxAB and LuxCDE, it's possible you can create
"Chimeric Proteins" of these two sets that will still work. Sometimes,
protein chimeras work at a higher efficiency, because the product of one
protein in the chain is ejected in close proximity to the active site of
the next protein in the chain.

To make protein chimeras, simply fuse the CDSs, removing the STOP codon
of the first protein and replacing it with a "spacer stretch" of fairly
boring/neutral, hydrophilic amino acids. Then surround this hybrid CDS
with a Promoter/RBS + Terminator as if it were only one protein.

If all goes well, the two sub-proteins will fold correctly, joined by a
"boring" spacer of inert amino acids, and will work as intended. The
benefit: You've saved a lot of promoter/Terminator DNA. It's even
possible that metabolic chains such as LuxCDE will work even better as
fusions.

On the other hand, the connection of three distinct proteins, each with
their own methods of self-folding, may result in a protein folding
train-wreck which utterly fails to work. In which case, far from saving
money spent on DNA, you've lost all the money spent on your protein
fusion. It's a gamble!

On 09/06/12 17:05, Mega wrote:
> Thought it would be best to start a new thread for this.
>
> I had the light producing genes (Lux ABCDE) rewritten by a program to adapt

> it to *Arabidopsis *(Codon Bias).

>
>
> Now I have to add promoter(s), RBS and terminator.
>
> According to this
> http://bccm.belspo.be/db/lmbp_plasmid_details.php?NM=pJE202 , in the native
> plasmid there is only one promoter "Vibrio fischeri luxICDABEG operon
> promoter".
> Translating this into plant genes, I just need one promotor for all the
> genes?? (Virus-Plant promoter, because of strong expression)
>
> When I attach the promoter sequence to the gene segence, does there have to
> be sapce left or just  add it like ATGTC + AAAA = ATGTCAAAA ??
>
>
> I think this would be the best promoter available: CLCuMV C1. Does anyone
> know where to get the sequence from??
>
>
> thx
>

--

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