Transposon Design

[Mega]

Hi, 

I may get the chance to get to work with a transposon, 

however I have a design question: 

Do I need to include a terminator? Because the DNA is inserted anywhere in the E.Coli genome, and somewhere afterwards there is a terminator for sure. 

For the basical design I'd ligate three PCR products together, and the terminator would be the fourth fragment to attach -> can I save the working time? :D  

Would you just go on without? 

Best,

[Cathal Garvey]

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If you don't add a terminator, you could be activating or silencing
(by transcribing mRNA reverse compliments) all kinds of genes in the
locality of your inserted transposon; this could have huge knock-on
effects for the cell, and consequently for your desired DNA's behaviour.

So, always include a terminator. :)

A bi-directional terminator would be a bonus, too, to make sure your
DNA isn't read into by some external promoters on the + or - strand.

If you had space, you could even put your favourite bi-directional
terminator on both ends of your transposon casette to fully insulate
your genes from read-through to either end.

That way, if your only clone happens to have your transposon shoved in
the middle of an active operon, at least it'll stop the operon from
interfering with your cassette (even if the messed-up-operon may have
downstream effects that *do* mess things up..).

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[Mega]

ok, thanks...

But the transposon will integrate at many different sites, so one may disrupt the cells metabolism, while another does not. Obviously one survives, while the other won't.

Terminators on both ends? Won`t that be prone to homologous recombination sooner or later?

[Cathal Garvey]

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> But the transposon will integrate at many different sites, so one
> may disrupt the cells metabolism, while another does not. Obviously
> one survives, while the other won't.

Not all interruptions will kill the cell, but some may mess with your
project. For example, say you want to use the Lac operator to control
your gene in response to lactose, but you accidentally read backwards
through the lac operon and silence LacZ, which is required to make
allolactose from lactose to activate the operator (phew). Now you've
got an integrated, stable transformant, which can survive but which
won't work correctly.

That's an obvious but highly unlikely example; far more likely is that
you mess up something more subtle and it doesn't show up until much
later; perhaps a stress response regulator or the like.

Obviously given that it's 2013, you'll be sequencing your inserts
anyway, but insulating your DNA is just the tidy thing to do. Keep
your code self-contained and you won't have to worry about outside
interference.

> Terminators on both ends? Won`t that be prone to homologous
> recombination sooner or later?

If they're big enough and identical, sure. They needn't be identical,
though; even if you used the "same" terminator, you could swap out
most of the nucleotides in the stem portion of the stem-loop for other
nucleotides as long as they all match up and have roughly the same
melting temperature. So they could be (shamelessly written in fastac
format: https://gitorious.org/fastac/fastac ):
~~~~~~~~~~~~~~~~~~
# Parameterised Stem-loop Section
{0}
CAG
{1}
$def_template stem_loop

> First_Terminator
; three-frame stop
TAAATAAATAAATAAA
; Stemloop template filled in with "CGAT" x 3 and rev-complement.
$use_template stem_loop -r "CGATCGATCGAT" "ATCGATCGATCG"
; Ribosomal Slow-Down Section
TTTATTTATTTATTTA

> Second_Terminator
; three-frame stop
TAAATAAATAAATAAA
; Stemloop template filled in with "GATC"
$use_template stem_loop -r "GATCGATCGATC" "GATCGATCGATC"
; Ribosomal Slow-Down Section
TTTATTTATTTATTTA
~~~~~~~~~~~~~~~~~~

...giving you:

> First_Terminator
taaataaataaataaacgatcgatcgatcagatcgatcgatcgtttattt
atttattta

> Second_Terminator
taaataaataaataaagatcgatcgatccaggatcgatcgatctttattt
atttattta

I don't actually advise you to use these terminators, I'm only
offering an example of how you could use the same basic terminator
format with differing stemloops and melting temperatures. They're not
guaranteed to be bidirectional, either; that's a dark art I
temporarily learned some time ago but have mostly forgotten at this
moment.

- -Cathal

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