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Judging/Variance/Johns Hopkins-BAG
From 2009.igem.org
Dear iGEM Team,
I am writing to you on behalf of the JHU BAG iGEM team to request a variance from the BioBrick standard of biological parts and assembly of biological parts. Our iGEM team has focused on developing new technologies and standards for synthetic genome construction. Our method for assembling genomic pieces depends on a new standard we refer to as the “Building Block”. The workflow of our system is as follows:
- Optimized protocols to create “Building Blocks” (BBs) by Overlap Extension/Assembly PCR from oligomers.
- USER (uracil specific excision reaction) protocol to join BBs into smaller chunks
- Homologous recombination in vivo to assemble smaller chunks to larger chunks, and using a second round of homologous recombination to assemble larger chunks into chromosome arms and full chromosomes
The purpose of our team is to develop and optimize techniques for large DNA construction as well as further the effort of the construction a synthetic Saccharomyces cerevisiae. Our team also has designed several novel computer programs for high throughput analysis of trace files and other sequence information. We fully understand the uniqueness of our team and we will be emphasizing that our central goals are somewhat distinct from those of most iGEM Teams (we are building a complex chassis, not stringing together parts into devices). All our methods will be described on our wiki from this perspective.
We hope that our team can provide new tools, both wet and dry, to broaden the perspective of iGEM and
provide a generic toolset for the synthetic Biology community.
Advantages of our Building Block Standard:
- Unlike BioBricks - No scars; seamless construction. Scars are not consistent with our genome redesign strategy because our knowledge of the rules of genome structure and function is insufficient.
- DNA sequence requirements at joints are extremely flexible and can be found in any sequence; consensus of joining sites is A {Nx} T where x= 3,5,7,9 or 11
- Much larger DNA pieces such as whole chromosomes may be assembled, with no constraints on restriction enzyme site positions.
- Building blocks that are assembled can more easily contain more parts (gene, promoter, rbs, etc.), and systems of parts of very different sizes
Disadvantages:
- Lack of standard Biobrick assembly standard
Difference from BioBricks:
- Building blocks used for genome assembly, may contain more than one part or may containing partial pieces of parts. This can be viewed as an advantage or a disadvantage, depending on perspective.
Respectfully submitted,
James DiCarlo,
JHU - BAG Team
