[BBF Standards] Fwd: new assembly standard proposal

[Drew Endy]

(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> 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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