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Genome engineering: new codes, new AAs, multi-virus resistance 2-3:30pm, CLSB 521 7-Nov-2012 SB204. Thanks to: .gov || || .edu || || .org || || .com || || Read = = = = = = = = I/O = = = = = = = Write. NHGRI. NIGMS. LSRF. ArmRev.org. Oppenheimer Foundation. Azco. - PowerPoint PPT PresentationTRANSCRIPT
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1
Genome engineering: new codes, new AAs, multi-virus resistance 2-3:30pm, CLSB 521 7-Nov-2012 SB204
Azco
ArmRev.org
Oppenheimer Foundation
Gen9
LSRF
NHGRINIGMS
A revolution in reading &
writing DNA
A terabyte per bite.
4
1944 1995 2008
55
What is a minimal replicating system ?
& why should we want one?
66
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).
77
What is a minimal replicating system ?
1999 Science: Nonessential Mycoplasma genitalium protein-coding genes: 130 of 482 (+43 tRNAs)2006 PNAS: 67 confirmed. 34 added
26: disrupted only in M. pneumoniae37: mixed mutant pools in liquid culture34: Limited sampling
88
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.
12
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.
doubling)
Reconstituted ribosomes:
Jewett & Church
1313
Translation
E P A
23+5S : 50S
16S : 30S
Safe Industrial Organisms Genome Engineering Rationale
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
22
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)
Commercial Systems: Roche, Ambion, Novagen, Promega, Invitrogen, Qiagen, Stratagene, Paragon, Amersham, NEB, Sutro, EMerck
Tony Forster(Vanderbilt)
23
Mirror world :
A highly flexible tRNAacylation method fornon-natural polypeptideSynthesis. Suga lab Nature Methods 2006
Construction of Modified Ribosomes forIncorporation of d-Amino Acids into Proteins. Hecht lab Biochemistry 2006
2424
A vesicle bioreactor as a step toward an artificialcell assembly --Noireaux & Libchaber PNAS
2004
-hemolysin-eGFP in vesicleeGFP no vesicle
Genetically encoded unnatural amino acidsLiu & Schultz 2010 Ann Rev Biochem.
4. Orthogonal antibiotics(ideally inexpensive)
e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) higher serum stability
Azide Strained cyclo-octyne
+
+
Prescher, JA & CR Bertozzi (2005) Nature Chem Biol. Chemistry in living systems
hydrazideKetone
triazole
hydrazone
Orthogonal AA chemistry (metabolic dependence)
e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) higher serum stability
AzidePhosphine
+
+
Prescher, JA & CR Bertozzi (2005) Nature Chem Biol. Chemistry in living systems
hydroxylamineKetone
Amide
Oxime
2828
10X /year since 2005 (vs 1.5X for VLSI)
Carr & Church, Nature Biotech
Amplify pools with flanking universal primers
6 Paths to error correction1.Hyb-Select: Tian et al. 2004 Nature 2. MutS: Carr & Jacobson 2004 NAR 3. MutHLS: Smith & Modrich 1997 PNAS4. Endo/Exonuclease : Bang Nat Meth. 20085. Errase 6. Sequencing
Minimum: $500 per 1M oligos
4 Next-Gen Synthesis: on chips
8K Xeotron Photo-Generated Acid
12K Combimatrix Electrolytic
120K Roche, Febit Photolabile 5'protection
244K Agilent Ink-jet standard reagents
29
OLS bp/error SynBIOSISKosuri et al Nature Biotech
200-mer 250 130-mer 1300 130+Errase 5940
30
PCRsort&
select
pick&
place
Oligo elute amplify
Data
Oligonucleotide-Design
Roche/454
Array synthesis
Agilent OLSPolonator
multiwell plate
PicoTitrePlate
Polonatorflowcell
Rolony Photorelease
"perfect" part
2 ways to Coalesce Next-Gen DNA reading & writing
Write Read Select
micromirror
Matzas, Church, et al. (Febit,HMS) Nat. Biotech Nov 2010
31
3232
Multiplex Automated Genome Engineering
(MAGE)
Integration of genome reading & writing
Micromirror-Polonator
Wang
Terry
33
#2: ds-Linear x Circle1 step 5’>3’exo Reda/E b/T + SelectZhang et al Nat.Gen 1998 Yu et al. PNAS 2000 (GeneBridges license)
#1: ds-Circle x Circle2 step recA+ recombination
+ Select + counterselectLink et al J. Bact 1997
(Open-access)
#3: ss-90mer x ds-Circle #4: ss-Mb x ds-Circle conjugation
Costantino &Court PNAS’03 Wang et al., Nature '09
4 DNA homology-directed strategiespKO3
E.coli
MAGE
CAGE
Isaacs et al., Science ‘11
8 Optimizations for ss allele replacement
Oligo length 10x
Phosphorothioate 3x
G > –13 kcal/mol 30x
[oligo] = 0.05-50uM
Wang, et al. Nature 2009 Isaacs et al Science 2011Ellis et al PNAS 2001
Red-10,000xMutS 100x
Lagging strand: 30x
Co-selection 4x
Co-Selection: another factor of 4
Samereplichore
Locus that restores antibiotic resistance
Cross replichore
-Red ssDNA Allelic Replacement
insertion
deletion
site-specific
mismatch
37
MAGEPrimase & Nuclease
Chris
Gregg
Josh
MosbergMarc
Lajoie
5 changes per 2h4E9 genomes/day/vial
30462
22705
11512
11802
12210
9620
18664
18894
22037
16795
7016
8797
314
2765
20899
1249
15082
15272
72898
5266
9540
7490
32080
11569
17791
13399
39835
21121
28866
30530
7401
4810
41644
34568
5733
38167
12119
32265
19820
9452
24106
29581
14174
4611611924
22067
1496
2771
24991
21050
36108
14901
20813
35252
46524
27567
44217
26270
24629
5443133875
40846
15115
10774
IsaacsCharalelChurchSunWang CarrJacobsonKong Sterling
New translation code: novel AA
Safety features: no functional DNA
exchange
multi-virus resistance
39
Why genome engineering? Multi-virus resistance
Changing 13/64 codons: ACC(T), AGA(R), AGG(R), ATA(I), CCC(P), CGG(R), CTC(L), CTT(L), GCC(A), GTC(V), TAG(-), TCC(S), TGA(-)
Isaacs, Lajoie, MosbergKosuri, Wang,Carr,et al
Clinical tests of non-standard AA “orthogonal” chemistry
PEG-pAcPhe-hGH Ambrx, Cho, Schultz et al. higher serum stability
+hydrazideKetone hydrazone
4141
Improving a Natural Enzyme Activity through Incorporation ofUnnatural Amino Acids -- Ugwumba et al 2010 JACS
8-11-fold improvement … in contrast to … screening hundreds of thousands of mutants with natural amino acids.
4 Selection technologies
In vitro
GC-MSClones
Sensor-selectors
42
4 ways for in vivo coupled sensors-selectors
3. ds-DNA 4. mRNA binding
2. tRNA-ribosome1. riboswitches
43
44
68 Sensor-Selectors (old & new ligands)
Vatsan Raman
56 DNA binding proteins: ada araC arcA argPR carP cpxR crp cspA cynR cysB cytR deoR dnaA dgsA fadR farR fhlA flhCD fnr fruR fur galR gcvA glpR hipB iclR ilvY lacI lexA lrp malT marR melR metJ metR modE nagC narL narP ntrC ompR oxyR pdhR phoB purR rbsR rhaS rpoE rpoH rpoN rpoS soxS tetR torR trpR tyrR12 Riboswitches: Adenine B12 FMN Guanine Glucosamine-6-phosphate Glycine di-GMP Lysine Molybdenum PreQ1 SAM SAH TPP theophylline 3-methylxanthine
4545
.
4646
.