Judging/Variance/BIOTEC Dresden

From 2009.igem.org

Team Request

Dear iGEM judges,

I am writing on behalf of the BIOTEC_Dresden team to kindly ask for your permission to use full DNA synthesis in conjunction with an alternative DNA assembly method, two custom bacterial plasmid backbones, and one custom fosmid backbone.

Full DNA synthesis and alternative DNA assembly method: In the spirit of next generation synthetic biology our team decided to make extensive use of iGEM sponsor Geneart's gene synthesis discount and have all our 7 required DNA constructs (almost 6 kb) synthesized. Since the traditional BioBrick assembly method, which produces a mixed XbaI / SpeI site ("BioBrick scar"), is unidirectional and can not be undone to separate assembled parts again, there is no reason to include "alibi" BioBrick scars between adjacent functional parts in the sequence of our DNA constructs. Therefore we omitted them and hope this is OK with you. To be able to provide those constructs to others through the BioBrick registry and allow for compatibility with other BioBricks, however, we did include flanking prefixes and suffixes. Full synthesis of DNA constructs does not only make BioBrick assembly obsolete, it also offers the opportunity for the transfer of those constructs to a desired plasmid backbone by homologous recombination ("Recombineering"), which is superior to conventional cut-and-ligate cloning in many respects. It is extremely efficient, does not require restriction enzymes or ligases, and it lacks the DNA size limitation of traditional ligations. Therefore we kindly ask for your permission to use this technology as an alternative DNA assembly method for plasmid and fosmid DNA manipulation. Recombineering by Red/ET homologous recombination in E.coli was first described by Zhang et al. (1998) and Muyrers et al. (1999).

Two custom bacterial plasmid backbones: In order to make direct use of existing Flp(F70L) expression plasmids for our purposes without transferring Flp to a BioBrick plasmid backbone, we would like to register two such existing plasmids with the BioBrick registry and provide them in a BioBrick-ready format with prefix and suffix. Both Flp expression plasmids will be the host backbone for one of our synthesized DNA constructs, for which we kindly ask your permission. These plasmids are based on pSC101 and contain an operon for anhydrotetracycline or L-rhamnose inducible Flp(F70L) expression, respectively. They were described by Sarov et al. (2003).

Fosmid: Furthermore, we would like to use a fosmid in our project because it offers the advantage of being present at only 1 copy per cell. The fosmid of choice is pCC2Fos from EPICENTRE Biotechnologies (http://www.epibio.com/item.asp?ID=385). We plan to use one such pCC2Fos vector that already contains a 39 kb insert from the human MECP2 genomic locus. We will provide this fosmid in BioBrick format to the registry and we kindly ask for your permission to use it as recipient backbone for 6 of our 7 synthesized DNA constructs, which we will all provide as BioBricks cloned into pSB1AK3 also.

We will add backbone sequences as BioBricks to the registry for both pSC101-Flp(F70L) plasmids and the pCC2Fos-MECP2 fosmid as soon as possible.

References: Muyrers JP, Zhang Y, Testa G, Stewart AF. Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res. 1999 Mar 15;27(6):1555-7.

Sarov M, Schneider S, Pozniakovski A, Roguev A, Ernst S, Zhang Y, Hyman AA, Stewart AF. A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nat Methods. 2006 Oct;3(10):839-44.

Zhang Y, Buchholz F, Muyrers JP, Stewart AF. A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet. 1998 Oct;20(2):123-8.

Please do not hesitate to inquire further. Thank you very much indeed!

With very best wishes from Dresden, Germany. The BIOTEC_Dresden Team