[BBF Standards] Fwd: new assembly standard proposal

Thu Jul 10 22:10:56 EDT 2008

Sure,

We've been doing a lot of playing around with assembly protocols.  The 1-2-3
method and modified 1-2-3 method were ones we were doing about a year ago.
We have only implemented them for BBb.  dbbs has been used sporadically with
both BBa and BBb--it's really good, but labor intensive.

The 1-2-3 method starts with methylating the 5' parent part (the lefty part)
and the 3' parent part (the righty part) in engineered coli strains called
lefty and righty that express methyltransferases.  Lefty methylates at
BglII, righty methylates at BamHI.  Other than this, the 1-2-3 method is
basically a suffix or prefix insertion reaction.  You make a cocktail of
BglII, BamHI, and XhoI in buffer, dispense it out, then add both the lefty
and righty parts.  That's the "1" of 1-2-3.  You then heat kill the
XhoI--that's the "2", then you add ligase and more BamHI and BglII, benchtop
ligate, and transform into lefty or righty for the next round--that's "3".

The modified 1-2-3 method adds a gel purification in lieu of step 2.

The basic 1-2-3 has variability issues giving anywhere between 10:1 or 1:10
desired product : parent vector.  The modified 1-2-3 method is really clean,
but the gel purification isn't easily amenable to automation.

The dbbs method involves ipcr using one of the oligos you'd use to sequence
biobrick parts (such as G00101) and an oligo that anneals somewhere on the
vector--we usually used one overlapping the AlwNI site.  You pcr off the
lefty and righty parts,do a cleanup, digest AlwNI/BamHI/DpnI for the lefty
part, and AlwNI/BglII/DpnI for the righty part, do a cleanup, ligate,
transform.  It's usually really clean, but the pcring introduces the
potential of all the usual issues one has with pcr-based methods--point
mutations, funky alternative products, recombination events.  Also, it is
really labor intensive.

Lately I've been inching towards "one-pot" methods, but they aren't ready
for prime time.

-Chris

On Thu, Jul 10, 2008 at 1:59 PM, Raik Gruenberg <raik.gruenberg at crg.es>
wrote:

> Hi Chris,
>
> could you please provide some link to a description of the different
> assembly methods you mentioned? Especially the 1-2-3 method pops up on iGem
> team web sites and your excellent OOW tutorials but I couldn't find any
> actual description of it.
>
> Greetings,
> Raik
>
> Drew Endy wrote:
>
>> (Forwarding a great reply from Chris Anderson at Berkeley to the full
>> standards list; for everybody's consideration and additional discussion).
>>
>>  Hey Tom,
>>>
>>> That's an interesting proposal, and I do think we are still in an era in
>>> which exploring different assembly schemes is prudent, and possibly
>>> necessary.
>>>
>>>
>>> First of all, there is the question of whether hybrid enzyme standards
>>> like Raik's make more or less sense than single pair standards.  My
>>> impression is that the hybrid strategy only exists to avoid re-making the
>>> collection of parts but serves no specific function beyond that.  There
>>> aren't yet enough basic parts in existence that moving them to a new
>>> standard would be a substantial effort.  Therefore this strikes me as a weak
>>> motivator, and your proposal of trying a new standard that would be
>>> fundamentally incompatible with BBa suggests to me you see it the same way.
>>>  Nevertheless, the EcoRI-AgeI-part-NgoMIV-PstI could be a standard, and I'll
>>> pretend for the sake of argument that this is what Raik proposed (and the
>>> existing parts would of course remain compatible with such a standard).
>>>
>>>
>>> In evaluating these things, it seems there are two distinct criteria
>>> categories to consider.  First is the acceptability of the scar sequence,
>>> and the second is the subtle effects of the enzymes on assembly chemistry.
>>>
>>>
>>> In terms of broad acceptance outside of iGEM, I think there will always
>>> be holdouts on the idempotent standard assembly concept as long as the scar
>>> is not scarless.  I have never seen a chemistry providing a scarless
>>> solution to this that would be robust enough to serve as the backbone of the
>>> long-term assembly solution (not for a lack of looking, and kudos to Austin
>>> Che for trying!)  I think ultimately we can get there, but it will require
>>> some pretty difficult protein engineering.  In the meantime, we have a
>>> variety of 6 bp scar options that give reasonable scars for protein
>>> fusions--the Silver lab standard, BglII/BamHI, AgeI/NgoMIV, and SpeI/AvrII
>>> (and there may be other reasonable ones).  The peptides they encode all seem
>>> arguably reasonable to me, so this does not seem to be a viable criterion
>>> for distinguishing the standards.
>>>
>>>
>>> The second criterion is the ease of assembly (including side reactions
>>> and frequent complications).  In practice this is very hard to evaluate as
>>> you must take into account the particular reaction scheme (prefix/suffix
>>> insertions, 3ab method, 1-2-3 method, one-pot method, dbbs (an ipcr scheme),
>>> the Goler PCR scheme, etc.) and the enzymes as they perform in that scheme.
>>>  The criteria you fault BglII/BamHI for--heat insensitivity and
>>> proliferation of genome fragments--both strike me as criteria predicated on
>>> a 3ab assembly scheme.  I would agree with you that BBb enzymes perform
>>> poorly in 3ab reactions.  However, the 1-2-3, one-pot, and pcr-based schemes
>>> of BBb do not involve heat killing or 3-part ligations.  In the case of the
>>> 1-2-3 and one-pot methods, the reactions involve site-specific methylation
>>> and background subtraction with the assembly enzymes.  So, the dominant
>>> issues are the performance of the cognate methyltransferases expressed from
>>> the coli genome and the overall efficiency of cutting-to-completion by the
>>> enzymes.  It is on these criteria that BglII/BamHI stands out.  I've never
>>> used NgoMIV, but I have worked with AvrII, AgeI, and of course SpeI and
>>> XbaI.  The reactivity of AvrII and SpeI are definitely better than AgeI and
>>> XbaI.
>>>
>>>
>>> One also has to evaluate these standards for the ease of assembly for
>>> both the short term in the pre-automation era, their perfomance in the
>>> soon-to-come automation-with-reagents era, the reagent-free assembly era,
>>> the scarless era, and potentially a later phage-based or cell-free era.  How
>>> well, for example, does NgoMIV express and perform in an in vitro
>>> transcription/translation mixture?  It may seem premature to consider such
>>> things now, but this is the inevitable path of development for idempotent
>>> assembly schemes and these issues will ultimately determine what assembly
>>> chemistries remain in use 5 years from now.
>>>
>>>
>>> One also must consider the course of development of oligonucleotide-based
>>> total synthesis that evolves in parallel to assembly schemes (and I won't
>>> even get into the implications of SLIC derivative methods).  If something
>>> dramatic happens in the market in the next 3 years bringing the price down
>>> two orders of magnitude, then this is all a big waste of brain energy.  If
>>> the total synthesis cost comes down at least to the point where pcr-based
>>> cloning of basic parts is impractical relative to total synthesis, then
>>> complications such as the frequency of internal restriction sites are
>>> irrelevant.  Of course, that day may also never happen and restriction site
>>> frequency may still be a concern 10 years from now.
>>>
>>>
>>> ...so, I think this is all pretty complicated.  I think about it a great
>>> deal, and my guestimations of the future lead me to BglII/BamHI.  However,
>>> it is all sufficiently complicated that the criteria that would lead away
>>> from BBb may be more relevant than my guestimations suggest, and I therefore
>>> think that having "felt out" a variety of alternative assembly chemistries
>>> is of great value.  They help guarantee that we avoid the worst case
>>> scenario--that we lock ourselves into the status quo and find ourselves at
>>> the same place we are now 5 years out.
>>>
>>>  -Chris
>>>
>>>
>>> On Tue, Jul 8, 2008 at 12:34 PM, Tom Knight <tk at csail.mit.edu <mailto:
>>> tk at csail.mit.edu>> wrote:
>>>
>>>
>>>
>>>
>>>
>>>
>>> --
>>> J. Christopher Anderson, Ph.D.
>>> Assistant Professor
>>> Department of Bioengineering
>>> http://andersonlab.qb3.berkeley.edu/
>>>
>>> Office: 308A Stanley Hall
>>> Lab: 327 Stanley Hall
>>>
>>> Mailing Address:
>>> J. Christopher Anderson
>>> University of California, Berkeley
>>> 327 Stanley Hall, Mailcode #1762
>>> Berkeley, CA 94720
>>>
>>
>>
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>>
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>
> --
> ________________________________
>
> Dr. Raik Gruenberg
> http://www.raiks.de/contact.html
> ________________________________
>

-- 
J. Christopher Anderson, Ph.D.
Assistant Professor
Department of Bioengineering
http://andersonlab.qb3.berkeley.edu/

Office: 308A Stanley Hall
Lab: 327 Stanley Hall

Office Phone: 510-666-3611
Lab Phone: 510-664-4200
Fax Line: 510-664-4200

Mailing Address:
J. Christopher Anderson
University of California, Berkeley
327 Stanley Hall, Mailcode #1762
Berkeley, CA 94720
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