Cloning of shine-dalgarno or kozak to CDS sans scar/gap/space

[Sebastian Cocioba]

Say you have a CDS and you want to clone in a different shine dalgarno or kozak consensus. How would you do the properly? Kozak and SD both require direct "contact" with the ATG start codon so only blunt cloning may work since just one base extra between consensus and the ATG and the kozak fails or dramatically drops expression. Its such a small fragment that you would have to PCR it into your sequence but what if your gene is large? Do you just amplify one reasonable chunk and then overlap PCR the rest? It seems like fidelity may play an important role if the gene of interest is huge. Maybe if the promoter is shorter, add it to that and Gibson the whole deal? Gibson is way too expensive for large experiments...especially for the home lab approach. In parts I narrowed the reagents for Gibson Assembly to $7/rxn and that's after asking for lab startup discounts, shopping around, and accounting for shipping. I wish there was a way to cheaply glue bits in the order I want without scars. Im sure someone on the list is a black belt in molecular bio and knows of some magical elixir to do so...or im just out if the loop with all the latest and most hip techniques and its blindingly obvious. :P

Any ideas for a good way to add a few tens of bases to genes effectively? I know there are myriad ways to add bits...just curious if anyone has preference or just pros/cons? Thanks!

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D

[Cory Tobin]

I would just make a primer that contains the modified ribosome binding
sequence at the 5' end. So the primer would match 100% to the CDS but
then have your extra bit just upstream of the ATG. When you PCR the
CDS using that primer you will get a fragment with your ribosome
binding sequence stuck to the end. If this won't work in your
situation there are some other methods I might recommend but none work
as reliably as PCR.

But you mentioned the CDS is large so you may not be able to clone the
whole thing at once. How big are we talking here? If it's 15kb or
less it shouldn't be a problem for a decent enzyme.

-cory

[Mega [Andreas Stuermer]]

That surely is the best bet for small genes.

How about this: Use a restriction site that contains ATG? But you would also need PCR to introduce it.

Or a nice restriction site in front of the gene? So your primer is nnnnnggtaccATGnnnnnnn? The a 3 bp before atg seems to be the most important part. gccaccATG is consensus, you might get nice expression levels with GgtACCATG also?

[xmort]

Dne sobota, 25. ledna 2014 16:36:44 UTC+1 Mega [Andreas Stuermer] napsal(a):

That surely is the best bet for small genes.

How about this: Use a restriction site that contains ATG? But you would also need PCR to introduce it.

Or a nice restriction site in front of the gene? So your primer is nnnnnggtaccATGnnnnnnn? The a 3 bp before atg seems to be the most important part. gccaccATG is consensus, you might get nice expression levels with GgtACCATG also?

The problem with the restriction site (here NcoI) is, that  it also changes the first aminoacid residue after the initial Met because it introduces G as the first base of second triplet. Also there might be another Nco somewhere  in the CDS. Thus it might or might not work well enough with the intended CDS. I would really go the PCR way, it is easy enough and works almost every time. It can also be done in paralel with plenty of genes, providing you have enough money for primers. If the cds is really large ~ 10kb plus, you can always do it "per partes" using naturaly occuring unique restriction sites.