Winston Retreat

1
S. cerevisiae 2.0

• Winston Retreat
• June 2006

2

• Engineered Biological Systems
• Nature has optimized biology (artifacts)
• Technologies exist to optimize differently
• Try to re-engineer

3

• Engineered Biological Systems
• Nature has optimized biology (artifacts)
• Technologies exist to optimize differently
• Try to re-engineer

so human use-able

• Of interest to biologists (test models)
• chemists (atomic control of living systems)
• technologists (biomaterials, energy, medicine)
• re-writers

4
From D. Endy
5
Previous Page
Sequence (BNL)
Dunn Studier, J. Mol. Bio. 166477 (1983)
From D. Endy
6
Wild-Type T7 Genes 2.8-3 ----------------2.8-----
------------gt acgcaaagggaggcgacatggcaggttacggcgcta
aaggaatccgaaa lt--3-RBS---gtlt----------------3-
-------------
From D. Endy
7
Wild-Type T7 Genes 2.8-3 ----------------2.8-----
------------gt acgcaaagggaggcgacatggcaggttacggcgcta
aaggaatccgaaa lt--3-RBS---gtlt----------------3-
-------------
T7.1 Parts 28 29 acgcaaGgggagAcgacaCggcaggttacg
gcgctaaggatccggccgcaaagggaggcgacatggcaggttacggcgct
aaa ----------------2.8-----------------gtltD28RD29
Lgtlt--3RBS------gtlt---------------3----
From D. Endy
8
Genome design algorithm
T7 39,937 bps 57 putative RBSs encoding 60
proteins 51 regulatory elements T7.1 41,326
bps 73 parts
From S. Kosuri, D. Endy
9
http//parts.mit.edu/
10
Refactor1-12,179T7
Wild-Type T7 (T7)
From D. Endy
11

• Two yeast rewrites
• mtDNA re-org
• SAGA swap

1. http//www.mbg.cornell.edu/MBG_Faculty_Detail
.cfm?id10
2. from Suzanne Berger to NAS 05/15/06
12
mtDNA re-org
mt DNA 85,779 bps 8 verified protein encoding
genes 24 tRNA genes 2 rRNA genes 20 nucleic acid
processing factors encoded by introns
http//db.yeastgenome.org/cgi-bin/gbrowse/yeast/?n
amechrMito3A1..85779
13
mtDNA re-org
COX1, ATP8, ATP6, COB, OLI1, VAR1, COX2, COX3,
8 proteins (7 for ox phos, 1 mt ribosome)
11 dubious ORFs
http//db.yeastgenome.org/cgi-bin/gbrowse/yeast/?n
amechrMito3A1..85779
14
mtDNA re-org
15S rRNA
21S rRNA
intron encodes I-Sce enzyme
http//db.yeastgenome.org/cgi-bin/gbrowse/yeast/?n
amechrMito3A1..85779
15
mtDNA re-org
Design 2.0
8 protein ORFs
2 rRNAs
25 tRNAs
reduces genome by 4.7 kb

• Design 3.0 might also remove introns
• reduces genome by 20.5 kb
• lose 20 nucleic acid modifiers
• might regulate with T7 RNAP instead of RPO41 and
  MTF1

16
mtDNA re-org
Execution 2.0
http//images.google.com/imgres?imgurlhttp//tran
splant.sinica.edu.tw/data/pds.jpgimgrefurlhttp/
/transplant.sinica.edu.tw/data/pds.htmlh323w30
7sz33tbnid9iaDZP47jhgGYMtbnh114tbnw108hl
enstart1prev/images3Fq3DPDS1000/He26svnum
3D1026hl3Den26lr3D26sa3DN
17
SAGA swap
Ada1 Ada2 Ada3 Gcn5
Spt3 Spt7 Spt8 Spt20
Taf5 Taf6 Taf9 Taf10 Taf12
Tra1
Sgf73 Sgf29 Sgf11 Ubp8 Sus1
from Wu Mol Cell (2004) 15199
18
SAGA swap essentl genes?
Ada1 Ada2 Ada3 Gcn5
Spt3 Spt7 Spt8 Spt20
Taf5 Taf6 Taf9 Taf10 Taf12
Tra1
Sgf73 Sgf29 Sgf11 Ubp8 Sus1
from Wu Mol Cell (2004) 15199
19
SAGA swap HAT?
Ada1 Ada2 Ada3 Gcn5
Spt3 Spt7 Spt8 Spt20
Taf5 Taf6 Taf9 Taf10 Taf12
Tra1
Sgf73 Sgf29 Sgf11 Ubp8 Sus1
from Wu Mol Cell (2004) 15199
20
SAGA swap HATTBPreg?
Ada1 Ada2 Ada3 Gcn5
Spt3 Spt7 Spt8 Spt20
Taf5 Taf6 Taf9 Taf10 Taf12
Tra1
Sgf73 Sgf29 Sgf11 Ubp8 Sus1
from Wu Mol Cell (2004) 15199
21
SAGA swap txn reg?
Ada1 Ada2 Ada3 Gcn5
Spt3 Spt7 Spt8 Spt20
Taf5 Taf6 Taf9 Taf10 Taf12
Tra1
Sgf73 Sgf29 Sgf11 Ubp8 Sus1
from Wu Mol Cell (2004) 15199
22
SAGA swap HATneighbor?
Ada1 Ada2 Ada3 Gcn5
Spt3 Spt7 Spt8 Spt20
Taf5 Taf6 Taf9 Taf10 Taf12
Tra1
Sgf73 Sgf29 Sgf11 Ubp8 Sus1
from Wu Mol Cell (2004) 15199
23
SAGA swap core subunits?
Ada1 Ada2 Ada3 Gcn5
Spt3 Spt7 Spt8 Spt20
Taf5 Taf6 Taf9 Taf10 Taf12
Tra1
Sgf73 Sgf29 Sgf11 Ubp8 Sus1
from Wu Mol Cell (2004) 15199
24
SAGA swap
Ada1 Ada2 Ada3 Gcn5
Spt3 Spt7 Spt8 Spt20
Taf5 Taf6 Taf9 Taf10 Taf12
Tra1
Sgf73 Sgf29 Sgf11 Ubp8 Sus1
from Wu Mol Cell (2004) 15199
25
(No Transcript)
26
the end
Break something