Dear iGEM Judges,
I’m writing on behalf of the 2008 UCSF iGEM Team to request permission to submit non-biobrick parts to the registry for this year’s Jamboree.
For the second year in a row, the UCSF iGEM team is using a homegrown combinatorial cloning technique for high efficiency, multipart ligations. The key to this approach is a Type II-S restriction enzyme (AarI) that can generate four base overhangs of any sequence. Digestion yields parts with standard, non-palindromic, overhangs.
Typically, we ligate a promoter and DNA parts encoding two different protein domains into an expression vector. This four-part ligation technique allows us to "shuffle" protein domain combinations/configuration and titrate expression levels with a variety of promoters. In our discussion with other teams, we determined that while biobricks are excellent for iterative construction protocols, four part ligations are not feasible. In addition, AarI cloning can be “scar-less”, which was necessary this year for a construct that would not tolerate amino-terminal extensions.
The AarI combinatorial cloning approach is founded in our experience that it is not always possible to predict what the most effective domain combination or expression level will be. This is particularly true for our current project, which involves harnessing heterochromatin as a synthetic control mechanism. There is little prior work in this area, and we needed to sample a large number of constructs quickly to identify effective fusion constructs.
In the spirit of iGEM, we hope to share these parts with the community, and to facilitate this transfer, we are constructing a shuttle plasmid for conversion of AarI parts into bio-brick parts. We will submit this vector with our parts, and describe the AarI cloning and conversion process on our wiki. Given the differing strengths of the AarI shuffle and Bio-Brick standards, we hope you will accept our constructs for this year's competition.
Thank you for your consideration,
Andrew Horwitz (Lim Lab)
UCSF iGEM Team
On behalf of the 2008 iGEM judging committee, thanks for your email and request granted.
As you write, please be sure that the team clearly documents the custom assembly method via a section in your final online materials and, if appropriate, via a note on your poster or in your presentation. For example, if you have the described shuttle plasmid system working, I'm sure that many people would be interested in learning about it.
Please also contact Randy Rettberg to ensure that any AarI-based parts contributed to the Registry (either as information in the database, or as physical material) are clearly marked as such and are expected.
Beyond iGEM, the broader BioBricks standards community would also likely be interested in learning about your experiences (standards AT biobricks DOT org).