[BBF Standards] housecleaning for physical assembly

[Raik Gruenberg]

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
________________________________