[BBF Standards] Fwd: new assembly standard proposal
Raik Gruenberg
raik.gruenberg at crg.es
Sun Jul 20 09:55:51 EDT 2008
Dear BioBrickers,
let's please try to unlock the current stalemate of follow-up BioBrick assembly
formats and proposals! I've reworked the discussion page on openwetware:
http://openwetware.org/wiki/The_BioBricks_Foundation:Standards/Technical/Formats
* Tom's proposal is now included in the list.
* The former sequence images are now in plain text to allow copy/pasting and
error correction.
More importantly, I've tried to compile the different arguments raised into a
single table that compares the different formats side-by-side:
http://tinyurl.com/5ch4qa
Please, feel free to correct and add more criteria for comparison! Please
complain if you think your method of choice was unfairly treated.
Greetings,
Raik
J. Christopher Anderson wrote:
> 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
> <mailto: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>
> <mailto: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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>
> --
> ________________________________
>
> 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
--
________________________________
Dr. Raik Gruenberg
http://www.raiks.de/contact.html
________________________________
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