Turn a gene around after PCR

[Mega]

Hi guys, 

When I do a PCR and then get an operon which is reversly orientated (see picture http://www.ncbi.nlm.nih.gov/gene/3280765 LuxA-G-fre needed) and I need to clone it next to a normally oriented promoter, what can I do to turn it around? 

I thouht about cutting with restriction enzymes, which will cut it in a way that they just fit together if correctly inserted. But none of the RE have restriction sites that I immagine would fit together... 

Has anyone experience with this? 

[Cathal Garvey]

Hey Mega,
DNA is bi-directional, or "antiparallel" as they like to say in
encyclopaedias. As a result, your DNA doesn't need to be "reversed", as
all genes run in the 5' to 3' direction: if you've got a gene on a
piece of DNA, one end is innately the end from which the gene is
interpreted, regardless of how it's presented in a genome viewer.

However, if you just add this to a promoter-bearing bit of DNA and add
ligase, you'll get 50% of your resulting clones in the backwards
orientation, with the promoter directing transcription from the end of
your gene. This would be a great way to knock-out a gene if that's what
you wanted..but I know it's not.

To prevent this from happening, you can either A) Cut your DNA with
different enzymes at either end, and PCR/cut your promoter-bearing DNA
with the same enzymes in the correct orientation, or B) Use PCR to
assemble your DNA prior to circularlising it.

To achieve A):
1. Repeat the PCR with new primers to add appropriate restriction sites
(you could add the biobrick prefix/suffix for example), remembering to
leave at least 2 nucleotides to either end of your restriction sites
(so-called "landing sites" for enzymes that require a bit more room
than their target sequence).
2. Do the same for your promoter-bearing plasmid or whatever: PCR the
whole thing with a proofreading polymerase with primers that have
additional enzyme sites added in the correct orientation.. again,
biobrick suffix/prefix will do: the suffix should be after the
promoter, so you can attach it to the prefix right before your CDS.
3. Cut both sequences.
4. Ligate both sequences.

To achieve B):
1. Get primers for your CDS that have additional 5' bits matching the
3' end of your promoter; 20n would be ideal, so your promoter looks
like 5'--20nPromoter-20nCDS--3'. The 3' promoter need not have an
overlap; you can circularlise it with ligase once it's correctly joined
at the other end.
2. PCR up your CDS again with these primers.
3. PCR your promoter-bearing element with primers that create a
matching 3' end where you want the two to overlap.
4. Mix some of the two PCR products and run another few rounds of PCR:
this will generate long fusions where the PCR'd promoter-region
"primes" the CDS from the overlap.
5. Dilute this PCR product, and run another round of PCR using the
forward primer for the promoter-bearing bit and the reverse primer for
the CDS.
6. You should now have *mostly* the full construct (confirm on a gel).
To circularise it, you need to just add ligase, but if it's too
concentrated, it's more likely to just form long chains of units joined
end on end. So, you need to dilute it first, so that when ligase binds
a free end and joins it to something, the nearest thing is the other
end of the same molecule! Perhaps someone else here recalls what
concentration range is best for this part?

Hope that helps,
Cathal

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[Andreas Sturm]

Wait,


all genes go at the 5' -> 3' strand (clearly)  and:  always in 5' -> 3' direction?


I got a bit confused why the genome viewers then show them to be in reverse orientation  "ATG-Gene-TAG" is then not  "GAT-eneG-GTA" on the top strand??

[Andreas Sturm]

Ah I found it out!! At least I think so!!!

The 3' -> 5' has the correct sequence in the wrong orientation but it's correct if you read it in 5' -> 3'. This one is the template for the PCRs. Correct?

So I can just design the primers for this one and then it's like doing a PCR from 5' -> 3' strand

[Nathan McCorkle]

Mega, why not just rotate the image in a photoshop-type program, then it will be in the normal orientation! It really is that simple though, you're just looking at things rotated around! Design your primers so either end has a different restriction sequence, then when you PCR the promoter just use the same restriction site for the end of the promoter.

Also make sure the ATG of the gene remains in the correct coding frame as the promoter, the distance from the start of the promoter to the start of the gene should be evenly divisible by 3

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--
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[Cathal Garvey]

Hey Nathan, Mega..
Not sure that the frame of the promoter matters so much; it's OK to have
some 5' bits of variable length AFAIK. Rather, the frame of the start
codon (and to a lesser extent the Kozak/Shine-Dalgarno sequence) that
matters.

I'd err on slightly more distance from promoter to start codon than
less, as transcription start points aren't always immediately at the
promoter itself, and may be poorly characterised. Perhaps even variable,
I can't claim to know..

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[Andreas Sturm]

Yeah, thank you... Figured that out, should have beed ds-DNA view then I'd have seen it earlier.

Well, AFAIK, the polymerase moves over the DNA strand until  - no you're correct. the RBS should have a certain distance from the ATG. However, in chloroplasts that doesn't matter because they seem not to need RBS   :D
 
For pUC19 insertion that may matter. pVIB is based on pBR322, so you've got 20 copies per cell. With pUC19 you'll get 100 copies per cell (IIRC). Immagine that glow ;)

[Cathal Garvey]

Bacteria (and therefore chloroplasts) do have ribosome binding sites,
but they are called "Shine Dalgarno" rather than "Kozak" consensus
sequences. Depending on the species they can be ~6-10n upstream of the
actual start codon; a variance of 1-2n won't kill expression but there
is an ideal gap.

The shine dalgarno sequence at least partially matches the 3' end of the
16S rRNA ribosomal subunit, so if you don't have an example of a
chloroplast shine-dalgarno sequence, you can cheat by copying the end of
a 16S transcript..but that can backfire, because a shine-dalgarno that's
too strong can actually prevent translation.

You should be able to find literature on chloroplast shine-dalgarnos,
I'd expect.

[Andreas Sturm]

http://www.ncbi.nlm.nih.gov/pubmed/9520261

Chlamydomonas reinhardtii chloroplasts don't need SD-sequences it seems. 

I remember to have read that it's the same with higher plants. That's quite logical, becuase ct-genomes are generally conserved quite well.  

[Andreas Sturm]

Yeah, it's even written in the abstract. 

[Cathal Garvey]

Neat! Makes life easier that way. E.coli is also pretty promiscuous
with start codons, and can apparently get away without a shine-dalgarno
in transgenes. I figured that was down to domestication and selection
for easy transformants, apparently not! :)

On Thu 22 Nov 2012 14:17:22 GMT, Andreas Sturm wrote:
> Yeah, it's even written in the abstract.
>