Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Genomics:GTL Program Projects
J. Craig Venter Institute
43
Progress toward a Synthetic Cellular Genome
Hamilton O. Smith* (hsmith@venterinstitute.org), Cynthia Pfannkoch, Holly A. Baden-Tillson, Clyde A. Hutchison III, and J. Craig Venter
J. Craig Venter Institute, Rockville, MD
To test our understanding of the genetic requirements for cellular life, we proposed to construct a minimal cellular genome by chemical synthesis (Hutchison, et al., 1999). A number of technical hurdles remain before this can be accomplished and we report progress on these here.
We improved upon the methodology and shortened the time required for accurate assembly of 5- to 6-kb segments of DNA from synthetic oligonucleotides. We first tested our methodology by assembly of infectious phiX174 genomes (Smith, et al., 2003). The methods have since been tested by assembling three segments (4.6, 5.3, and 6.5 kb) of the mouse mitochondrial genome. In each case assembly was straightforward, so we feel the methods are quite robust. We have made improvements in these assembly methods aimed at improving sequence accuracy of the assembled DNA.
About one lethal error per 500 bp occurred in our phiX174 genome assembly, assuming a random distribution of errors among assembled genomes. We have approached the elimination of such errors at four levels: 1) Purifying oligonucleotides prior to assembly by high-throughput capillary electrophoresis, to remove molecules containing errors that lead to mutations 2) Modifying assembly conditions to minimize DNA damage leading to mutations, 3) Global error correction of the assembled product by biochemical methods directed at producing the consensus sequence of the assembled product, and 4) Efficient correction of errors by oligonucleotide-directed mutagenesis following cloning and sequencing of the assembled product.
To drive development of improved methodology we are undertaking some larger synthetic projects. We are assembling the complete genome of the mouse mitochondrion. We are also synthesizing a region from the M. genitalium genome that encodes genes essential for translation of mRNA to produce proteins.
* Presenting author
