Terminators in reverse
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Sebastian Cocioba
Jul 19, 2014, 1:41:46 AM7/19/14
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Quick question:
Would a terminator sequence work if cloned in reverse?
I have primers for amplifying the nopaline synthase terminator but in my new vector the two cutters in question are in the reverse order. If I clone it in, it would be "antisense" to the proper sequence. Since terminators, in general, are just hairpin physical structures, would the reverse still work in eukaryotes?
Im making a combinatorial set of my transient expression vector for use in testing my gene gun using various promoters and terminators and so it would be great if I can save some money on primers and explore the effects of a reverse terminator on overall expression rate. Just wanted to see if anyone has some experience before I waste the few bucks on cloning the terminator in. Thanks!
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
Would a terminator sequence work if cloned in reverse?
I have primers for amplifying the nopaline synthase terminator but in my new vector the two cutters in question are in the reverse order. If I clone it in, it would be "antisense" to the proper sequence. Since terminators, in general, are just hairpin physical structures, would the reverse still work in eukaryotes?
Im making a combinatorial set of my transient expression vector for use in testing my gene gun using various promoters and terminators and so it would be great if I can save some money on primers and explore the effects of a reverse terminator on overall expression rate. Just wanted to see if anyone has some experience before I waste the few bucks on cloning the terminator in. Thanks!
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
Cathal Garvey
Jul 19, 2014, 6:42:55 AM7/19/14
to diy...@googlegroups.com
Depends on the terminator, actually. Some of them are bidirectional,
others are not.
Firstly, there are two kinds; rho-dependent and rho-independent. I don't
know enough to speculate about rho-dependent, so I won't. The
latter-however, are structural in nature, and work roughly like this:
The polymerase, upon meeting a terminator, transcribes it to RNA, but as
the RNA is transcribed it rapidly forms a secondary structure whose free
energy is high enough to sort of "yank out" the RNA strand,
destabilising the polymerase. For this to happen, the structure has to
form quickly, and usually the terminator contains a tract of nucleotides
immediately after it that slow down the polymerase, such as A/T rich DNA.
Now, imagine that the structure is forming as it is synthesised; there
is therefore a very slight directionality to the structure, particularly
at and around the hairpin. So, the difference between bidirectional and
mono-directional terminators can be very subtle.
When I was making my first IndieBB I made my own terminator based on
information in a paper, which I have since lost. I tried to make it
bidirectional, but the only way to be sure is to test it. :)
Anyways; where possible, when in doubt use the terminator in the
direction you know to work. It's hard to know or to predict
directionality, the empirical approach is best.
--
T: @onetruecathal, @IndieBBDNA
P: +353876363185
W: http://indiebiotech.com
others are not.
Firstly, there are two kinds; rho-dependent and rho-independent. I don't
know enough to speculate about rho-dependent, so I won't. The
latter-however, are structural in nature, and work roughly like this:
The polymerase, upon meeting a terminator, transcribes it to RNA, but as
the RNA is transcribed it rapidly forms a secondary structure whose free
energy is high enough to sort of "yank out" the RNA strand,
destabilising the polymerase. For this to happen, the structure has to
form quickly, and usually the terminator contains a tract of nucleotides
immediately after it that slow down the polymerase, such as A/T rich DNA.
Now, imagine that the structure is forming as it is synthesised; there
is therefore a very slight directionality to the structure, particularly
at and around the hairpin. So, the difference between bidirectional and
mono-directional terminators can be very subtle.
When I was making my first IndieBB I made my own terminator based on
information in a paper, which I have since lost. I tried to make it
bidirectional, but the only way to be sure is to test it. :)
Anyways; where possible, when in doubt use the terminator in the
direction you know to work. It's hard to know or to predict
directionality, the empirical approach is best.
T: @onetruecathal, @IndieBBDNA
P: +353876363185
W: http://indiebiotech.com
Sebastian Cocioba
Jul 19, 2014, 1:47:45 PM7/19/14
to diy...@googlegroups.com
Thanks for the info!
Seems there is still so much to learn. Time to dig up a ton of articles
on eukaryotic terminators. Ill post again on this thread if I find
anything worth note or answer my own question regarding Tnos. Its about
time I actually learn how these things work in detail. Seems my issue
has always been an obsession with technique at the expense of
theoretical understanding.
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D From: Cathal Garvey
Sent: 7/19/2014 6:42 AM
To: diy...@googlegroups.com
Subject: Re: [DIYbio] Terminators in reverse
Seems there is still so much to learn. Time to dig up a ton of articles
on eukaryotic terminators. Ill post again on this thread if I find
anything worth note or answer my own question regarding Tnos. Its about
time I actually learn how these things work in detail. Seems my issue
has always been an obsession with technique at the expense of
theoretical understanding.
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Sent: 7/19/2014 6:42 AM
To: diy...@googlegroups.com
Subject: Re: [DIYbio] Terminators in reverse
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