Team Freiburg Bioware
With three years of experience in iGEM and after last year's second place the iGEM Team Freiburg is highly motivated to deliver
some good piece of synthetic biology in 2009. In this year we want to
create an universal restriction enzyme to facilitate labwork
and enable new techniques.
We're looking forward to meeting you on this year's jamboree!
|Universal Endonuclease – Cutting
Gene technology is driven by the use of restriction endonucleases. Yet, constraints of limited sequence length and variation recognized by available restriction enzymes pose a major roadblock for synthetic biology. We developed the basis for universal restriction enzymes, primarily for routine cloning but also with potential for in vivo applications. We use a nucleotide cleavage domain fused to a binding domain, which recognizes a programmable adapter that mediates binding to DNA and thus cleavage. As adapter we use readily available modified oligonucleotides, as binding domain anticalins and as cleavage domain FokI moieties engineered for heterodimerization and activity. For cloning, this universal enzyme has merely to be mixed with the sequence specific oligonucleotide and the target DNA. Binding and release are addressed with thermocycling. Additionally, we provide concepts for in vivo applications by external adapter delivery, activity regulation by photo-switching, as well as for modifying an argonaute protein towards a DNA endonuclease.