[BBF Standards] housecleaning for physical assembly

[Drew Endy]

As background for this thread... Reshma Shetty just published a paper  
on some new BioBrick vectors (http://www.jbioleng.org/content/2/1/5).   
In designing and synthesizing the new vector parts she worked to  
remove many restriction endonuclease sites.  From her article...

"To adhere to the BioBrick standard for physical composition, BioBrick  
vector parts need only be free of the BioBrick restriction enzyme  
sites. However, we chose to design anew all BioBrick vector parts  
(Figure 5), so that we could completely specify their DNA sequences.  
We compiled a list of potentially useful endonuclease sites for  
removal from all new BioBrick vector parts (Table 1). We targeted each  
group of endonuclease sites for removal for a different reason. We  
targeted recognition sites of enzymes that produce compatible cohesive  
ends to the BioBrick enzymes because such enzymes often prove useful  
in constructing new variants of BioBrick vectors. We targeted offset  
cutter sites because they may be useful in alternative restriction  
enzyme-based assembly methods [57]. We targeted homing endonuclease  
sites because they are commonly used in genome engineering [58]. We  
targeted some nicking endonuclease sites because they can be useful  
for specialized cloning applications [59]. Finally, we targeted  
several additional restriction endonuclease sites to keep them  
available for use by new standards for physical composition. Our list  
of endonuclease sites constitutes a set of target sequences that  
should be considered for removal from any newly synthesized BioBrick  
part, if possible. The target sequence set will change as the  
synthetic biology community develops new standards for physical  
composition of BioBrick parts. Some of the targeted endonuclease sites  
were naturally absent from the DNA sequences encoding our new vector  
parts. For any remaining sites, we removed the recognition sequences  
from the BioBrick vector parts by introducing point mutations.  
However, the functions of the pSC101 and pUC19-derived plasmid  
replication origins were sensitive to introduced mutations, so the  
replication origins used in this work are not free of all targeted  
endonuclease sites (see Methods). Similarly, issues during synthesis  
led to an unnecessary redesign of the ccdB positive selection marker,  
so it too is not free of all targeted endonuclease sites."

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On May 3, 2008, at 6:53 PM, Drew Endy wrote:
> 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
>> ________________________________
>
>
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