Bio Bricks
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Mega
Jun 2, 2012, 11:51:16 AM6/2/12
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Once again I came over the term biobrick...
I know what it should mean (Promotor is one brick, gene another, origin of replication , etc.)
But when you put them together, why are you sure that they will have the right place (1-2-3-4) instead of sticking together anyhow (in a 4-2-1-3 manner) ??
Do they have speciel restriction sites?? That do not attach to itself?
If I understood correctly, one can assemble plasmids (or even entire genoms) using this technique. And if the promoter doesn't work, just take aonther biobrick-promotor. And then it may work.
Maybe a stupid question for one who is familiar with it...
I know what it should mean (Promotor is one brick, gene another, origin of replication , etc.)
But when you put them together, why are you sure that they will have the right place (1-2-3-4) instead of sticking together anyhow (in a 4-2-1-3 manner) ??
Do they have speciel restriction sites?? That do not attach to itself?
If I understood correctly, one can assemble plasmids (or even entire genoms) using this technique. And if the promoter doesn't work, just take aonther biobrick-promotor. And then it may work.
Maybe a stupid question for one who is familiar with it...
Cory Tobin
Jun 2, 2012, 1:25:20 PM6/2/12
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Have a look at this page http://partsregistry.org/Assembly:Standard_assembly
If you look at the final product shown in the picture you will see
that the restriction sites in between parts get destroyed while the
two pairs of sites on either end of the combined part still exist. So
in any BioBrick compatible plasmid you only have 4 restriction sites,
no matter how parts you have combined into one. And the two ends of
the parts are different so you can specifically target which end you
want your insert to go to.
> If I understood correctly, one can assemble plasmids
> (or even entire genoms) using this technique. And if
> the promoter doesn't work, just take aonther
> biobrick-promotor. And then it may work.
Sort of. Once you assemble parts together there is no way to go back
and remove one part and replace it with another. You would need to
keep the all the plasmids you made up to that point, go back and find
the plasmid just before you added the part you want to swap, and use a
different part instead. Then rebuild the rest of the assembly. I'm
not sure I explained that clearly, if it is confusing let me know.
Since the BioBricks assembly doesn't let you swap out
already-assembled parts, the way people usually go about building
something big is they assemble a bunch of smaller components first,
test the small components, then start merging smaller components into
larger and larger ones until the whole thing is assembled. If you
build the whole thing at once and something doesn't work properly you
would need to rebuild the entire thing from the beginning.
-cory
If you look at the final product shown in the picture you will see
that the restriction sites in between parts get destroyed while the
two pairs of sites on either end of the combined part still exist. So
in any BioBrick compatible plasmid you only have 4 restriction sites,
no matter how parts you have combined into one. And the two ends of
the parts are different so you can specifically target which end you
want your insert to go to.
> If I understood correctly, one can assemble plasmids
> (or even entire genoms) using this technique. And if
> the promoter doesn't work, just take aonther
> biobrick-promotor. And then it may work.
and remove one part and replace it with another. You would need to
keep the all the plasmids you made up to that point, go back and find
the plasmid just before you added the part you want to swap, and use a
different part instead. Then rebuild the rest of the assembly. I'm
not sure I explained that clearly, if it is confusing let me know.
Since the BioBricks assembly doesn't let you swap out
already-assembled parts, the way people usually go about building
something big is they assemble a bunch of smaller components first,
test the small components, then start merging smaller components into
larger and larger ones until the whole thing is assembled. If you
build the whole thing at once and something doesn't work properly you
would need to rebuild the entire thing from the beginning.
-cory
Andreas Sturm
Jun 3, 2012, 4:02:39 AM6/3/12
to diy...@googlegroups.com
Ah, thanks!!
That made it more clear to me!
That made it more clear to me!
2012/6/2 Cory Tobin <cory....@gmail.com>
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Pieter
Jun 3, 2012, 6:17:42 AM6/3/12
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In theory the biobrick assembly is a very elegant method, in practice it is often a real pain. Especially when you try to use 3 way ligation for combining a big and a small brick, such as a large protein coding sequence and a small RBS. For the assembly of small bricks I would recommend overlapping PCR instead or when working with multiple parts go for Gibson assembly instead:
http://2010.igem.org/Team:Cambridge/Gibson/Introduction
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