Construction of a Bacterial Cell that Contains Only the Set of Essential Genes Necessary to Impart Life
Abstract
Work has continued on the Top Down approach to genome minimization. We have used the Essential (E), Non-essential (N), and Impaired (I) gene categories to make steady progress with gene and gene cluster deletions. To date, we have removed approximately 234 kb from the Mycoplasma mycoides JCVI-syn1.0 genome. The resultant 844 kb genome is viable and grows with a normal growth rate. The Bottom Up approach has also continued. New Tn5 gene disruption data allows a much more reliable classification of genes as E, I, or N. We have designed a new reduced genome design (RGD1) based on this data. Two initial segments from this 8 segment RGD1 design have been synthesized and are in testing for viability. We are also experimenting with an approach to construct RDG1 from PCR products amplified from syn1.0 genomic DNA. One 1/8th molecule is being synthesized by this PCR amplification method. The effort to modularize the genome is in progress. A tRNA module has been constructed and is currently being sequenced prior to testing for the ability to replace the natural tRNA genes. Preliminary work aimed at genome complementation has been conducted. A plasmid system for quickly adding back deleted genes will allow us to quickly examine which gene(s) within a deleted cluster might be causing growth defects or cell death. Experimental data using a two plasmid system suggests that development of a complementation system is possible.
Document Details
- Document Type
- Technical Report
- Publication Date
- Aug 16, 2013
- Accession Number
- ADA584428
Entities
People
- Anthony Yee
- Clyde Hutchison
- Hamilton Smith
- John I Glass
Organizations
- J. Craig Venter Institute