[BBF Standards] housecleaning for physical assembly

[Drew Endy]

Raik,

More great comments.  Note that your newly ordered set of parts aren't  
BioBrick parts (at least not yet, until we can get this standard  
issues settled).

What are other people thinking?  What other experiences do folks  
have?  Anybody from Berkeley want to chime in?  And, TK?

Thanks,
Drew

On May 3, 2008, at 6:36 PM, Raik Gruenberg wrote:

> Hi Drew, Hi Kim,
>
> thanks for this comment, this is indeed becoming an urgent topic!
> Obviously, I am endorsing the Freiburg Standard, not least because I  
> have already ordered a set of Biobricks in this standard ;-)
>
> The main advantage over the other suggestions out there is that it  
> delivers pretty much full compatibility to 1.0. That is, Freiburg  
> Expression parts behave like protein coding 1.0 parts and can as  
> such be combined with 1.0 promoters and RBS etc. without any  
> surprises.
>
> The downside is that it has so far only been used by one lab (the  
> Freiburg guys) who however tell me that they are still using it and  
> didn't come across any difficulties. They are not using 3A assembly  
> though (I will try out the 3A assembly as soon as my order arrives,  
> towards end of May.)
>
> Another issue is that real backwards compatibility is only given if  
> the Expression part indeed does not contain neither the 3.0 *nor*  
> the 1.0 restriction sites -- that means we have 6 rather than 4  
> forbidden sites.
>
> I am less convinced of organism-specific formats. Protein biobricks  
> can often be used unchanged in different organisms -- for example,  
> some gene synthesis companies routinely offer simultaneous yeast +  
> human codon optimization. The idea to minimize native restriction  
> sites is attractive but (1) we don't have that many choices of  
> enzyme pairs with compatible overhang and nice scar to start with  
> and (2) when manipulating one organism one may often want to use  
> (orthogonal) protein parts from another organism.
>
> A final possibility would be to produce a hybrid of the Freiburg and  
> the Berkeley format -- using the Freiburg compatibility prefix/ 
> suffix but replacing NgoMIV and AgeI by the Berkeley enzymes. If  
> Berkeley has kept the outer enzymes of 1.0, this format would have  
> the same compatibility advantage as the Expression part proposal.  
> Lacking documentation of the Berkeley format, I can't really verify  
> this. I was told the Berkeley scar is Gly-Ser, which is a routine  
> flexible linker for protein domains. Some may argue it is even too  
> flexible (forcefully helix breaking) but it is indeed very commonly  
> used.
>
> A last advantage of the Freiburg pair of inner enzymes is that there  
> is a blunt cutting isoschizomers for one of them. This means one can  
> excise a part and directionally transfer it into another format with  
> minimal added spacer. I'll try to transfer some of my Biobricks into  
> a Biofusion compatibility vector using this strategy (see last  
> section of the WIKI).
>
> Ah, and a very last little advantage: there is a Zinc finger library  
> out there that uses the same restriction sites because Zinc fingers  
> turn out to have Thr-Gly in their natural linker. Now this is a very  
> special-purpose advantage but two colleagues of mine are actually  
> quite thrilled about that and want to switch their Zinc finger  
> projects to Freiburg Biobrick cloning. (well, more generally this  
> example rather argues for a seamless Biobrick cloning strategy like  
> it seems to be possible with BB++).
>
> Greetings,
> Raik
>
>>>  I think it should obvious that the Biobr
> ick 1.0 standard is not
>>> what we want as it doesn't allow the construction of fusion   
>>> proteins.  The silver Biofusion standard has its merits, but I  
>>> fear  there may problems with the biochemistry of the scarring  
>>> site.  I  have successfully made functional fusion proteins with  
>>> it.  I also  believe that I'm having trouble with the scarring  
>>> site blocking a  short import tag in my project.  I'll know the  
>>> results of this in a  few weeks.
>>>
>>>  I like Raik's 3.0 Expression parts, although having longer tails   
>>> may lead to more difficult PCR's which could be a problem if you  
>>> are  trying to make a yeast gene knockout construct.  I also don't  
>>> like  the incompatibility idea, although whatever we choose should  
>>> be  implemented now before there are many more (yeast) Biobricks  
>>> in the  registry.  I'm going to make a naive suggestion: maybe an  
>>> organism  specific Biobrick system that utilizes the least  
>>> occurring sites  that can be made by fermentas?  This would save  
>>> time in one instance  and add much more in others...
>>>
>>>  And finally I don't agree with the idea of suggesting an  
>>> untested  standard.  How many times have engineers said "it looks  
>>> fine on  paper" only to discover the horrible truth out in the  
>>> real world?   Thats my 2 cents worth.  Cheers!
>>>
>>> Kim
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> -- 
> ________________________________
>
> Dr. Raik Gruenberg
> http://www.raiks.de/contact.html
> ________________________________