[BBF Standards] Tom Knight RFC (plain text)

Tue Jul 8 17:11:23 EDT 2008

Hi Tom,

your suggestion is very elegant because it retains the old overhang. Did you 
also have a look at
http://openwetware.org/wiki/The_BioBricks_Foundation:Standards/Technical/Formats
?

So, apart from the Silver lab and Berkeley suggestions there is a third proposal 
out there, which is the Expression Part aka "Freiburg format". Let me briefly 
recapitulate this format to complete your Background section. The Freiburg 
(actually Kristian M. Mueller and Katja M.Arndt) proposal looks like this:

EcoRI...XbaI-tg-NgoMIV <part> AgeI-taat-SpeI...PstI

This format introduces an additional restriction site pair rather than replacing 
the old one. The "scar" is ACCGCC producing Thr-Gly which can again be 
considered near optimal (the optimality of Gly-Ser is arguable anyway). This 
variant retains full backward compatibility to the classic format -- If the old 
assembly is used, the protein parts behave like stand-alone protein generators 
with start and stop. NgoMIV and AgeI can be heat-inactivated and the assembly 
has been tested by the Freiburg team.

Disclaimer:
I've gene-synthesized a list of about 50 protein fusion parts in this format and 
had planned to write up a RFC together with K. Mueller. However, we both have 
been slow at this. Right now, I am converting the 3A assembly vectors to the 
Freiburg format.

In any case, we quite urgently have to settle on a follow up standard and your 
suggestion looks very good too. For now, I just wanted to complete the record, 
so we basically have 4 proposals on the table: Silver lab, Berkeley, Freiburg, 
and yours.

Greetings,
Raik

Drew Endy wrote:
> Just a quick resend of Tom Knight's RFC as plain text.
> 
> ******
> 
> Request for comments
> 
> Biobrick assembly standard modifications
> 
> 8 July 2008
> 
> Tom Knight
> 
> 
> Background:
> 
> Over the past several years the original Biobrick assembly standard  
> has proven to be a useful DNA assembly technique.  Despite this, a  
> significant flaw has been the composition of the mixed base scar, T  
> ACTAGA G.  Since this scar is 8 bp long, it makes protein fusions,  
> aligned on three base codon boundaries, quite difficult.  Ira Phillips  
> at the Silver Lab worked around this problem by ignoring the flanking  
> T and G sites (inserted for protection against methylation issues) and  
> using the mixed site ACTAGA.  This resulted in the amino acid sequence  
> Thr-Arg inserted into fusion protein designs, two amino acids with  
> significant chemical difficulties in many contexts.
> 
> Chris Anderson at Berkeley worked around this problem in a different  
> way, by adopting a  new restriction enzyme set, BglII (prefix, AGATCT  
> site) and BamHI (suffix, GGATCC site).  These enzymes are insensitive  
> to methylation, and produce a scar GGATCT (Gly-Ser).  The Gly-Ser  
> amino acids are near ideal for most protein fusion work, and the  
> enzymes are cheap and effective.  Unfortunately, neither of these  
> enzymes can be heat inactivated, making automated assembly with them  
> substantially more difficult.
> 
> Another difficulty with these enzymes is the frequency of the BamHI  
> and BglII sites in many natural DNA sequences.  For example, in the E.  
> coli genome the BamHI average fragment length is 9,000, while the  
> average fragment length of XbaI fragment is 120,000.  This reflects  
> the relative rarity of the CTAG sequence in E. coli genomic DNA (for  
> reasons poorly understood).  The high frequency of sites causes two  
> problems.  First, making new Biobricks from existing genomic DNA  
> becomes substantially more difficult.  Second, the frequent occurrence  
> of these sites in contaminating genomic DNA in minipreps results in  
> short fragments which can replace desirable parts in assembly  
> reactions, yielding incorrect products.
> 
> Proposal:
> 
> Two additional restriction enzymes exist with a CTAG overhang: AvrII  
> (CCTAG site) and NheI (GCTAGC site).  AvrII cannot be heat killed, and  
> produces poorer codon choices than NheI.  I propose that we  
> restructure the cloning site and flanking sites of Biobrick parts with  
> the following structure:
> 
> ……<EcoRI>……<SpeI> Part <NheI>…..<PstI>…..
> 
> The part would be flanked by bare SpeI and NheI sites.  The mixed site  
> formed by assembly of these fragments, using standard approaches,  
> would be GCTAGT, coding for Ala-Ser.  The Ala-Ser amino acids are  
> almost as fusion-friendly as the Gly-Ser of the Anderson fusion  
> technique.
> 
> The NheI enzyme can be heat killed, and thus is more amenable to  
> automated assembly processes.
> 
> The rarity of the NheI site in E. coli genomic DNA means that many  
> fewer fragments accidentally cut from genomic DNA contamination of  
> minipreps will clone in place of the desired part.
> 
> Transition issues:
> 
> We would need to construct new cloning vectors with the new cloning  
> site.  Parts would need to be recloned into the new vectors, probably  
> using PCR with new primers.  Manual assembly of parts mixed between  
> old and new formats would likely be possible in many cases as an  
> interim solution, since the parts retain a common CTAG overhang.
> 
> We should rethink the use of the EcoRI enzyme for the prefix outside  
> cutter.  There are likely more robust enzymes usable.
> 
> We should rethink the need/desirability of the NotI sites between the  
> outside and inside restriction enzyme sites.  Some DNA fragment is  
> necessary there, but it need not be that sequence, and the two  
> sequences need not be identical.
> 
> Plan:
> 
> 1)    Circulate this document for comments and blunder stopping
> 
> 2)    Analyze the frequency of NheI sites in existing registry parts
> 
> 3)    Test the efficiency of NheI and any other recommended enzymes
> 
> 4)    Test for the ability to heat kill the enzymes
> 
> 5)    Design automated programs to assist in the primer design for  
> transition
> 
> 6)    Choose which parts are worth transitioning
> 
> 7)    Design desirable part collections for protein fusion work
> 
> 
>   
> _______________________________________________
> Standards mailing list
> Standards at biobricks.org
> http://biobricks.org/mailman/listinfo/standards_biobricks.org
> 

-- 
________________________________

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