[BBF Standards] Fwd: new assembly standard proposal

[Raik Gruenberg]

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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-- 
________________________________

Dr. Raik Gruenberg
http://www.raiks.de/contact.html
________________________________