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Genome engineering: new codes, new AAs, multi-virus resistance 2-3:30pm, CLSB 521 7-Nov-2012 SB204
Ribozyme-Catalyzed Transcription of an Active Ribozyme
Aniela Wochner, James Attwater, Alan Coulson, Philipp Holliger, Science April 2011. Evolution & engineering of an RNA polymerase ribozyme capable of synthesizing RNAs of up to 95 nt (& synthesis of a 27 nt hammerhead endonuclease ribozyme).
26: disrupted only in M. pneumoniae37: mixed mutant pools in liquid culture34: Limited sampling
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Essential genes of a minimal bacterium
PNAS 2006
99
A Whole-Cell Computational ModelPredicts Phenotype from Genotype
Karr et al. Cell 2012
1010
A Whole-Cell Computational ModelPredicts Phenotype from Genotype
Karr et al. Cell 2012
1111
Creation of a Bacterial Cell
Controlled by a Chemically
Synthesized Genome Gibson, et al 2010
Science
1 bp deletion in dnaA Not detected: Sanger 1-kb, but wrong clone sent by Blue Heron. At 10-kb level, because many of the 454 reads happened to end or start near error.
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113 kbp DNA 151 genes
Pure translation:Forster & Church
MSB ’05 GenomeRes.’06
Shimizu, Ueda ’01
Smaller.Higher speed &
accuracy requires a few extra genes(E.coli 20 min.
1. Robust, rapid doubling 10-20 minutes2. Non-standard amino acids3. Dependence on NSAAs4. DNA non-exchangeable with environment5. Multi-virus resistance
Testing essentiality of codons (13/64)
Codon Removed Natural Role # in Genome
# in 47 / 290 Essentials # Left
TAG Stop 322 0 0
AGA/AGG Arg 4,228 5 0CTT/CTC Leu 30,030 70 1*
CCC Pro 7,401 6 0ACC Thr 31,766 151 0ATA Ile 5,797 2 0GTC Val 20,757 67 0
GCC Ala 34,747 82 0TCC Ser 11,672 88 0CGG Arg 7273 6 0TGA Stop 1,232 4 0
Total 155,225 481 0 (Jul’12)
Green= 13 codon elimination Blue = 62 codon shuffle
Lajoie, Kosuri, Mosberg, Gregg, D.Zhang
One exception of 481 codons
ACT CTT GCC CTWG Vsr site highly specific mismatch repair
Lajoie, Kosuri, Mosberg, Gregg, D.Zhang
4 Mbp genome CAD
1919
Divide & Conquer Genome Engineering
2020
Complete Chemical Synthesis, Assembly,
and Cloning of a Mycoplasma genitalium
Genome -- Gibson, et al 2008 Science
Genome Engineering: full genome vs partial
1. E.coli > Yeast > Mycoplasma vs one species
2. Design & synthesis error consequences
3. 1E-7 genome transplantation efficiency
4. $1M vs $1 to $1k per Mb for raw DNA
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Applications of in vitro translation
•Ribosome display•Membrane protein drug receptor studies•Personal cancer vaccines.•Labeling one protein not the whole cell.•New chemistries (e.g. mirror chirality)