Computing the Future of Biology & Biotechnologyarchive2.cra.org/ccc/files/docs/Endy_CCC_lite.pdf · ~5E9 tons plants (1% DNA, made up number) ~1E9 tons animals (1% DNA, made up number)

Drew EndyStanford Department of BioengineeringThe BioBricks Foundation

9 March 2009

Computing the Future of Biology & Biotechnology

http://i.pbase.com/u47/hodero/large/30691408.jungle.jpg

Questions

What is this stuff?

Where did it come from?

How does it work?

What is it doing?

Where is it going?



Image c/o http://www.rps.psu.edu/probing/graphics/earth2.jpg

How much DNA on Earth?

http://www.rps.psu.edu/probing/graphics/earth2.jpg

http://www.rps.psu.edu/probing/graphics/earth2.jpg

~1E10 tons bacteria (5% DNA)~5E9 tons plants (1% DNA, made up number)~1E9 tons animals (1% DNA, made up number)~1E31 viruses (50% DNA)


The pangenome concept: a unifying view of genetic information.1: Med Sci Monit. 2005 Jul;11(7):HY24-9. Epub 2005 Jun 29.


4E35 bp bacterial DNA4E34 bp plant DNA1E34 bp animal DNA5E30 bp virus DNA

5E35 bp DNA on the planet

How quickly can we sequence?

1E10 bp per sequencing center per month

1E12 bp per month per Earth

5E23 months to sequence all DNA on earth

7Image c/o http://picasaweb.google.com/dipanjanbhattacharya/AroundMITD80#5213062475793120674

http://picasaweb.google.com/dipanjanbhattacharya/AroundMITD80#5213062475793120674

http://picasaweb.google.com/dipanjanbhattacharya/AroundMITD80#5213062475793120674

8Carlson, Pace & Proliferation of Biological Technologies, Biosec. & Bioterror. 1(3):1 (2003)

Typical NIH R01 c.2090

Specific Aim #1:

Sequence all DNA on earth in first month of project

Reviewer Comments:

Data storage & sharing plans not adequate

Functional annotation tools insufficient

10

Questions.

Can we finish genetics?

Could we finish early?

11

12

1. Genomes have finite lengths.

2. Fixation rates of mutants in populations are finite.

3. Atrophy rates of functional genetic elements are greater than zero.

Genetics is bounded?!

1. Collect or make libraries of mutants; characterize or screen.

13

Pre-Sequencing Genetics

2. Map mutants to loci.

Underlying Math = Logic

xxxxx xxxxxxxxx

xxx

xxx x

1. Read DNA.

14

Post-Sequencing Genetics

2. Identify putative functional genetic elements via models.

Underlying Math = Pattern Recognition

xxxxx xxxxxxxxx

xxx

xxx x

1. Design and construct any mutant or combinations thereof; characterize.

15

Post-Synthesis Genetics?

2. Reveal / confirm functions or demonstrate absence thereof.

Underlying Math =? Perturbation Design

Bacteriophage

16http://www.bact.wisc.edu/themicrobialworld/Phage.html

http://www.bact.wisc.edu/themicrobialworld/Phage.html

http://www.bact.wisc.edu/themicrobialworld/Phage.html

17

Key Features - self assembling - nanoscale - reproducing machine - programmed via DNA

Ecological importance

18

http://svs.gsfc.nasa.gov/vis/a000000/a002400/a002497/index.html



Technical importance

19

http://dspace.mit.edu/handle/1721.1/39677



20

Phage T7: Pre-Sequencing

“19 genes have been identified in T7 by isolation & characterization of amber mutants... ... it is estimated that not more than another 10 or so genes remain to be discovered.”

21

Phage T7: Post-Sequencing

“...coding sequences for 50 genes...”

“...evidence for expression of only 38...”

~100 genetic elements total

22

23

24

TCTCACAGTGTACGGACCTAAAGTTCCCCCATAGGGGGTACCTAAAGCCCAGCCAATCACCTAAAGTCAACCTTCGGTTGACCTTGAGGGTTCCCTAAGGGTTGGGGATGACCCTTGGGTTTGTCTTTGGGTGTTACCTTGAGTGTCTCTCTGTGTCCCTATCTGTTACAGTCTCCTAAAGTATCCTCCTAAAGTCACCTCCTAACGTCCATCCTAAAGCCAACACCTAAAGCCTACACCTAAAGACCCATCAAGTCAACGCCTATCTTAAAGTTTAAACATAAAGACCAGACCTAAAGACCA

GACCTAAAGACACTACATAAAGACCAGACCTAAAGACGCCTTGTTGTTAGCCATAAAGTGATAACCTTTAATCATTGTCTTTATTAATACAACTCACTATAAGGAGAGACAACTTAAAGAGACTTAAAAGATTAATTTAAAATTTATCAAAAAGAGTATTGACTTAAAGTCTAACCTATAGGATACTTACAGCCATCGAGAGGGACACGGCGAATAGCCATCCCAATCGACACCGGGGTCAACCGGATAAGTAGACAGCCTGATAAGTCGCACGAAAAACAGGTATTGACAACATGAAGTAACATGC

AGTAAGATACAAATCGCTAGGTAACACTAGCAGCGTCAACCGGGCGCACAGTGCCTTCTAGGTGACTTAAGCGCACCACGGCACATAAGGTGAAACAAAACGGTTGACAACATGAAGTAAACACGGTACGATGTACCACATGAAACGACAGTGAGTCACCACACTGAAAGGTGATGCGGTCTAACGAAACCTGACCTAAGACGCTCTTTAACAATCTGGTAAATAGCTCTTGAGTGCATGACTAGCGGATAACTCAAGGGTATCGCAAGGTGCCCTTTATGATATTCACTAATAACTGCACG

AGGTAACACAAGATGGCTATGTCTAACATGACTTACAACAACGTTTTCGACCACGCTTACGAAATGCTGAAAGAAAACATCCGTTATGATGACATCCGTGACACTGATGACCTGCACGATGCTATTCACATGGCTGCCGATAATGCAGTTCCGCACTACTACGCTGACATCTTTAGCGTAATGGCAAGTGAGGGCATTGACCTTGAGTTCGAAGACTCTGGTCTGATGCCTGACACCAAGGACGTAATCCGCATCCTGCAAGCGCGTATCTATGAGCAATTAACGATTGACCTCTGGGAAGAC

GCAGAAGACTTGCTCAATGAATACTTGGAGGAAGTCGAGGAGTACGAGGAGGATGAAGAGTAATGTCTACTACCAACGTGCAATACGGTCTGACCGCTCAAACTGTACTTTTCTATAGCGACATGGTGCGCTGTGGCTTTAACTGGTCACTCGCAATGGCACAGCTCAAAGAACTGTACGAAAACAACAAGGCAATAGCTTTAGAATCTGCTGAGTGATAGACTCAAGGTCGCTCCTAGCGAGTGGCCTTTATGATTATCACTTTACTTATGAGGGAGTAATGTATATGCTTACTATCGGTCTA

CTCACCGCTCTAGGTCTAGCTGTAGGTGCATCCTTTGGGAAGGCTTTAGGTGTAGCTGTAGGTTCCTACTTTACCGCTTGCATCATCATAGGAATCATCAAAGGGGCACTACGCAAATGATGAAGCACTACGTTATGCCAATCCACACGTCCAACGGGGCAACCGTATGTACACCTGATGGGTTCGCAATGAAACAACGAATCGAACGCCTTAAGCGTGAACTCCGCATTAACCGCAAGATTAACAAGATAGGTTCCGGCTATGACAGAACGCACTGATGGCTTAAAGAAAGGTTATATGCC

CAATGGCACACTATACGCTGCAAATCGGCGAATAGTGAGAACTTGGCGAGAGAACAACCTCGAACGCCGCAAGGACAAGAGAGGGCGGCGTGGCATAGACGAAAGGAAAAGGTTAAAGCCAAGAAACTCGCCGCACTTGAACAGGCACTAGCCAACACACTGAACGCTATCTCATAACGAACATAAAGGACACAATGCAATGAACATTACCGACATCATGAACGCTATCGACGCAATCAAAGCACTGCCAATCTGTGAACTTGACAAGCGTCAAGGTATGCTTATCGACTTACTGGTCG

AGATGGTCAACAGCGAGACGTGTGATGGCGAGCTAACCGAACTAAATCAGGCACTTGAGCATCAAGATTGGTGGACTACCTTGAAGTGTCTCACGGCTGACGCAGGGTTCAAGATGCTCGGTAATGGTCACTTCTCGGCTGCTTATAGTCACCCGCTGCTACCTAACAGAGTGATTAAGGTGGGCTTTAAGAAAGAGGATTCAGGCGCAGCCTATACCGCATTCTGCCGCATGTATCAGGGTCGTCCTGGTATCCCTAACGTCTACGATGTACAGCGCCACGCTGGATGCTATACGGTGG

TACTTGACGCACTTAAGGATTGCGAGCGTTTCAACAATGATGCCCATTATAAATACGCTGAGATTGCAAGCGACATCATTGATTGCAATTCGGATGAGCATGATGAGTTAACTGGATGGGATGGTGAGTTTGTTGAAACTTGTAAACTAATCCGCAAGTTCTTTGAGGGCATCGCCTCATTCGACATGCATAGCGGGAACATCATGTTCTCAAATGGAGACGTACCATACATCACCGACCCGGTATCATTCTCGCAGAAGAAAGACGGTGGCGCATTCAGCATCGACCCTGAGGAACTCATCA

AGGAAGTCGAGGAAGTCGCACGACAGAAAGAAATTGACCGCGCTAAGGCCCGTAAAGAACGTCACGAGGGGCGCTTAGAGGCACGCAGATTCAAACGTCGCAACCGCAAGGCACGTAAAGCACACAAAGCTAAGCGCGAAAGAATGCTTGCTGCGTGGCGATGGGCTGAACGTCAAGAACGGCGTAACCATGAGGTAGCTGTAGATGTACTAGGAAGAACCAATAACGCTATGCTCTGGGTCAACATGTTCTCTGGGGACTTTAAGGCGCTTGAGGAACGAATCGCGCTGCACTGG

CGTAATGCTGACCGGATGGCTATCGCTAATGGTCTTACGCTCAACATTGATAAGCAACTTGACGCAATGTTAATGGGCTGATAGTCTTATCTTACAGGTCATCTGCGGGTGGCCTGAATAGGTACGATTTACTAACTGGAAGAGGCACTAAATGAACACGATTAACATCGCTAAGAACGACTTCTCTGACATCGAACTGGCTGCTATCCCGTTCAACACTCTGGCTGACCATTACGGTGAGCGTTTAGCTCGCGAACAGTTGGCCCTTGAGCATGAGTCTTACGAGATGGGTGAAGCACGCTT

CCGCAAGATGTTTGAGCGTCAACTTAAAGCTGGTGAGGTTGCGGATAACGCTGCCGCCAAGCCTCTCATCACTACCCTACTCCCTAAGATGATTGCACGCATCAACGACTGGTTTGAGGAAGTGAAAGCTAAGCGCGGCAAGCGCCCGACAGCCTTCCAGTTCCTGCAAGAAATCAAGCCGGAAGCCGTAGCGTACATCACCATTAAGACCACTCTGGCTTGCCTAACCAGTGCTGACAATACAACCGTTCAGGCTGTAGCAAGCGCAATCGGTCGGGCCATTGAGGACGAGGCTCG

CTTCGGTCGTATCCGTGACCTTGAAGCTAAGCACTTCAAGAAAAACGTTGAGGAACAACTCAACAAGCGCGTAGGGCACGTCTACAAGAAAGCATTTATGCAAGTTGTCGAGGCTGACATGCTCTCTAAGGGTCTACTCGGTGGCGAGGCGTGGTCTTCGTGGCATAAGGAAGACTCTATTCATGTAGGAGTACGCTGCATCGAGATGCTCATTGAGTCAACCGGAATGGTTAGCTTACACCGCCAAAATGCTGGCGTAGTAGGTCAAGACTCTGAGACTATCGAACTCGCACCTGAATA

CGCTGAGGCTATCGCAACCCGTGCAGGTGCGCTGGCTGGCATCTCTCCGATGTTCCAACCTTGCGTAGTTCCTCCTAAGCCGTGGACTGGCATTACTGGTGGTGGCTATTGGGCTAACGGTCGTCGTCCTCTGGCGCTGGTGCGTACTCACAGTAAGAAAGCACTGATGCGCTACGAAGACGTTTACATGCCTGAGGTGTACAAAGCGATTAACATTGCGCAAAACACCGCATGGAAAATCAACAAGAAAGTCCTAGCGGTCGCCAACGTAATCACCAAGTGGAAGCATTGTCCGGTC

GAGGACATCCCTGCGATTGAGCGTGAAGAACTCCCGATGAAACCGGAAGACATCGACATGAATCCTGAGGCTCTCACCGCGTGGAAACGTGCTGCCGCTGCTGTGTACCGCAAGGACAAGGCTCGCAAGTCTCGCCGTATCAGCCTTGAGTTCATGCTTGAGCAAGCCAATAAGTTTGCTAACCATAAGGCCATCTGGTTCCCTTACAACATGGACTGGCGCGGTCGTGTTTACGCTGTGTCAATGTTCAACCCGCAAGGTAACGATATGACCAAAGGACTGCTTACGCTGGCGAAAG

GTAAACCAATCGGTAAGGAAGGTTACTACTGGCTGAAAATCCACGGTGCAAACTGTGCGGGTGTCGATAAGGTTCCGTTCCCTGAGCGCATCAAGTTCATTGAGGAAAACCACGAGAACATCATGGCTTGCGCTAAGTCTCCACTGGAGAACACTTGGTGGGCTGAGCAAGATTCTCCGTTCTGCTTCCTTGCGTTCTGCTTTGAGTACGCTGGGGTACAGCACCACGGCCTGAGCTATAACTGCTCCCTTCCGCTGGCGTTTGACGGGTCTTGCTCTGGCATCCAGCACTTCTCCGC

GATGCTCCGAGATGAGGTAGGTGGTCGCGCGGTTAACTTGCTTCCTAGTGAAACCGTTCAGGACATCTACGGGATTGTTGCTAAGAAAGTCAACGAGATTCTACAAGCAGACGCAATCAATGGGACCGATAACGAAGTAGTTACCGTGACCGATGAGAACACTGGTGAAATCTCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGCTGGTCAATGGCTGGCTTACGGTGTTACTCGCAGTGTGACTAAGCGTTCAGTCATGACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTC

AACAAGTGCTGGAAGATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCGAATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGGTGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTGGCTGCTGAGGTCAAAGATAAGAAGACTGGAGAGATTCTTCGCAAGCGTTGCGCTGTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGCCTATTCAGACGCGCTTG

AACCTGATGTTCCTCGGTCAGTTCCGCTTACAGCCTACCATTAACACCAACAAAGATAGCGAGATTGATGCACACAAACAGGAGTCTGGTATCGCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAGTGTGGGCACACGAGAAGTACGGAATCGAATCTTTTGCACTGATTCACGACTCCTTCGGTACCATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTGCGCGAAACTATGGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCA

GTTCGCTGACCAGTTGCACGAGTCTCAATTGGACAAAATGCCAGCACTTCCGGCTAAAGGTAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTAACGCCAAATCAATACGACTCACTATAGAGGGACAAACTCAAGGTCATTCGCAAGAGTGGCCTTTATGATTGACCTTCTTCCGGTTAATACGACTCACTATAGGAGAACCTTAAGGTTTAACTTTAAGACCCTTAAGTGTTAATTAGAGATTTAAATTAAAGAATTACTAAGAGAGGACTTTAAGTATGCGTAACTTC

GAAAAGATGACCAAACGTTCTAACCGTAATGCTCGTGACTTCGAGGCAACCAAAGGTCGCAAGTTGAATAAGACTAAGCGTGACCGCTCTCACAAGCGTAGCTGGGAGGGTCAGTAAGATGGGACGTTTATATAGTGGTAATCTGGCAGCATTCAAGGCAGCAACAAACAAGCTGTTCCAGTTAGACTTAGCGGTCATTTATGATGACTGGTATGATGCCTATACAAGAAAAGATTGCATACGGTTACGTATTGAGGACAGGAGTGGAAACCTGATTGATACTAGCACCTTCTACCACCACG

ACGAGGACGTTCTGTTCAATATGTGTACTGATTGGTTGAACCATATGTATGACCAGTTGAAGGACTGGAAGTAATACGACTCAGTATAGGGACAATGCTTAAGGTCGCTCTCTAGGAGTGGCCTTAGTCATTTAACCAATAGGAGATAAACATTATGATGAACATTAAGACTAACCCGTTTAAAGCCGTGTCTTTCGTAGAGTCTGCCATTAAGAAGGCTCTGGATAACGCTGGGTATCTTATCGCTGAAATCAAGTACGATGGTGTACGCGGGAACATCTGCGTAGACAATACTGCTAACAGTTA

CTGGCTCTCTCGTGTATCTAAAACGATTCCGGCACTGGAGCACTTAAACGGGTTTGATGTTCGCTGGAAGCGTCTACTGAACGATGACCGTTGCTTCTACAAAGATGGCTTTATGCTTGATGGGGAACTCATGGTCAAGGGCGTAGACTTTAACACAGGGTCCGGCCTACTGCGTACCAAATGGACTGACACGAAGAACCAAGAGTTCCATGAAGAGTTATTCGTTGAACCAATCCGTAAGAAAGATAAAGTTCCCTTTAAGCTGCACACTGGACACCTTCACATAAAACTGTACGCTATCC

TCCCGCTGCACATCGTGGAGTCTGGAGAAGACTGTGATGTCATGACGTTGCTCATGCAGGAACACGTTAAGAACATGCTGCCTCTGCTACAGGAATACTTCCCTGAAATCGAATGGCAAGCGGCTGAATCTTACGAGGTCTACGATATGGTAGAACTACAGCAACTGTACGAGCAGAAGCGAGCAGAAGGCCATGAGGGTCTCATTGTGAAAGACCCGATGTGTATCTATAAGCGCGGTAAGAAATCTGGCTGGTGGAAAATGAAACCTGAGAACGAAGCTGACGGTATCATTCAGGGTC

TGGTATGGGGTACAAAAGGTCTGGCTAATGAAGGTAAAGTGATTGGTTTTGAGGTGCTTCTTGAGAGTGGTCGTTTAGTTAACGCCACGAATATCTCTCGCGCCTTAATGGATGAGTTCACTGAGACAGTAAAAGAGGCCACCCTAAGTCAATGGGGATTCTTTAGCCCATACGGTATTGGCGACAACGATGCTTGTACTATTAACCCTTACGATGGCTGGGCGTGTCAAATTAGCTACATGGAGGAAACACCTGATGGCTCTTTGCGGCACCCATCGTTCGTAATGTTCCGTGGCACCGAG

GACAACCCTCAAGAGAAAATGTAATCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAAGGAGACACTTTATGTTTAAGAAGGTTGGTAAATTCCTTGCGGCTTTGGCAGCTATCCTGACGCTTGCGTATATTCTTGCGGTATACCCTCAAGTAGCACTAGTAGTAGTTGGCGCTTGTTACTTAGCGGCAGTGTGTGCTTGCGTGTGGAGTATAGTTAACTGGTAATACGACTCACTAAAGGAGGTACACACCATGATGTACTTAATGCCATTACTCATCGTCATTGTAGGATGCCTT

GCGCTCCACTGTAGCGATGATGATATGCCAGATGGTCACGCTTAATACGACTCACTAAAGGAGACACTATATGTTTCGACTTCATTACAACAAAAGCGTTAAGAATTTCACGGTTCGCCGTGCTGACCGTTCAATCGTATGTGCGAGCGAGCGCCGAGCTAAGATACCTCTTATTGGTAACACAGTTCCTTTGGCACCGAGCGTCCACATCATTATCACCCGTGGTGACTTTGAGAAAGCAATAGACAAGAAACGTCCGGTTCTTAGTGTGGCAGTGACCCGCTTCCCGTTCGTCCGTCTGT

TACTCAAACGAATCAAGGAGGTGTTCTGATGGGACTGTTAGATGGTGAAGCCTGGGAAAAAGAAAACCCGCCAGTACAAGCAACTGGGTGTATAGCTTGCTTAGAGAAAGATGACCGTTATCCACACACCTGTAACAAAGGAGCTAACGATATGACCGAACGTGAACAAGAGATGATCATTAAGTTGATAGACAATAATGAAGGTCGCCCAGATGATTTGAATGGCTGCGGTATTCTCTGCTCCAATGTCCCTTGCCACCTCTGCCCCGCAAATAACGATCAAAAGATAACCTTAGGTGAAAT

CCGAGCGATGGACCCACGTAAACCACATCTGAATAAACCTGAGGTAACTCCTACAGATGACCAGCCTTCCGCTGAGACAATCGAAGGTGTCACTAAGCCTTCCCACTACATGCTGTTTGACGACATTGAGGCTATCGAAGTGATTGCTCGTTCAATGACCGTTGAGCAGTTCAAGGGATACTGCTTCGGTAACATCTTAAAGTACAGACTACGTGCTGGTAAGAAGTCAGAGTTAGCGTACTTAGAGAAAGACCTAGCGAAAGCAGACTTCTATAAAGAACTCTTTGAGAAACATAAGGATAA

ATGTTATGCATAACTTCAAGTCAACCCCACCTGCCGACAGCCTATCTGATGACTTCACATCTTGCTCAGAGTGGTGCCGAAAGATGTGGGAAGAGACATTCGACGATGCGTACATCAAGCTGTATGAACTTTGGAAATCGAGAGGTCAATGACTATGTCAAACGTAAATACAGGTTCACTTAGTGTGGACAATAAGAAGTTTTGGGCTACCGTAGAGTCCTCGGAGCATTCCTTCGAGGTTCCAATCTACGCTGAGACCCTAGACGAAGCTCTGGAGTTAGCCGAATGGCAATACGTTCCGG

CTGGCTTTGAGGTTACTCGTGTGCGTCCTTGTGTAGCACCGAAGTAATACGACTCACTATTAGGGAAGACTCCCTCTGAGAAACCAAACGAAACCTAAAGGAGATTAACATTATGGCTAAGAAGATTTTCACCTCTGCGCTGGGTACCGCTGAACCTTACGCTTACATCGCCAAGCCGGACTACGGCAACGAAGAGCGTGGCTTTGGGAACCCTCGTGGTGTCTATAAAGTTGACCTGACTATTCCCAACAAAGACCCGCGCTGCCAGCGTATGGTCGATGAAATCGTGAAGTGTCACGA

AGAGGCTTATGCTGCTGCCGTTGAGGAATACGAAGCTAATCCACCTGCTGTAGCTCGTGGTAAGAAACCGCTGAAACCGTATGAGGGTGACATGCCGTTCTTCGATAACGGTGACGGTACGACTACCTTTAAGTTCAAATGCTACGCGTCTTTCCAAGACAAGAAGACCAAAGAGACCAAGCACATCAATCTGGTTGTGGTTGACTCAAAAGGTAAGAAGATGGAAGACGTTCCGATTATCGGTGGTGGCTCTAAGCTGAAAGTTAAATATTCTCTGGTTCCATACAAGTGGAACACTGCT

GTAGGTGCGAGCGTTAAGCTGCAACTGGAATCCGTGATGCTGGTCGAACTGGCTACCTTTGGTGGCGGTGAAGACGATTGGGCTGACGAAGTTGAAGAGAACGGCTATGTTGCCTCTGGTTCTGCCAAAGCGAGCAAACCACGCGACGAAGAAAGCTGGGACGAAGACGACGAAGAGTCCGAGGAAGCAGACGAAGACGGAGACTTCTAAGTGGAACTGCGGGAGAAAATCCTTGAGCGAATCAAGGTGACTTCCTCTGGGTGTTGGGAGTGGCAGGGCGCTACGAACAATAAA

GGGTACGGGCAGGTGTGGTGCAGCAATACCGGAAAGGTTGTCTACTGTCATCGCGTAATGTCTAATGCTCCGAAAGGTTCTACCGTCCTGCACTCCTGTGATAATCCATTATGTTGTAACCCTGAACACCTATCCATAGGAACTCCAAAAGAGAACTCCACTGACATGGTAAATAAGGGTCGCTCACACAAGGGGTATAAACTTTCAGACGAAGACGTAATGGCAATCATGGAGTCCAGCGAGTCCAATGTATCCTTAGCTCGCACCTATGGTGTCTCCCAACAGACTATTTGTGATATACGC

AAAGGGAGGCGACATGGCAGGTTACGGCGCTAAAGGAATCCGAAAGGTTGGAGCGTTTCGCTCTGGCCTAGAGGACAAGGTTTCAAAGCAGTTGGAATCAAAAGGTATTAAATTCGAGTATGAAGAGTGGAAAGTGCCTTATGTAATTCCGGCGAGCAATCACACTTACACTCCAGACTTCTTACTTCCAAACGGTATATTCGTTGAGACAAAGGGTCTGTGGGAAAGCGATGATAGAAAGAAGCACTTATTAATTAGGGAGCAGCACCCCGAGCTAGACATCCGTATTGTCTTCTCAAGC

TCACGTACTAAGTTATACAAAGGTTCTCCAACGTCTTATGGAGAGTTCTGCGAAAAGCATGGTATTAAGTTCGCTGATAAACTGATACCTGCTGAGTGGATAAAGGAACCCAAGAAGGAGGTCCCCTTTGATAGATTAAAAAGGAAAGGAGGAAAGAAATAATGGCTCGTGTACAGTTTAAACAACGTGAATCTACTGACGCAATCTTTGTTCACTGCTCGGCTACCAAGCCAAGTCAGAATGTTGGTGTCCGTGAGATTCGCCAGTGGCACAAAGAGCAGGGTTGGCTCGATGTGGGATAC

CACTTTATCATCAAGCGAGACGGTACTGTGGAGGCAGGACGAGATGAGATGGCTGTAGGCTCTCACGCTAAGGGTTACAACCACAACTCTATCGGCGTCTGCCTTGTTGGTGGTATCGACGATAAAGGTAAGTTCGACGCTAACTTTACGCCAGCCCAAATGCAATCCCTTCGCTCACTGCTTGTCACACTGCTGGCTAAGTACGAAGGCGCTGTGCTTCGCGCCCATCATGAGGTGGCGCCGAAGGCTTGCCCTTCGTTCGACCTTAAGCGTTGGTGGGAGAAGAACGAACTGGTCA

CTTCTGACCGTGGATAATTAATTGAACTCACTAAAGGGAGACCACAGCGGTTTCCCTTTGTTCGCATTGGAGGTCAAATAATGCGCAAGTCTTATAAACAATTCTATAAGGCTCCGAGGAGGCATATCCAAGTGTGGGAGGCAGCCAATGGGCCTATACCAAAAGGTTATTATATAGACCACATTGACGGCAATCCACTCAACGACGCCTTAGACAATCTCCGTCTGGCTCTCCCAAAAGAAAACTCATGGAACATGAAGACTCCAAAGAGCAATACCTCAGGACTAAAGGGACTGAGTTGGA

GCAAGGAAAGGGAGATGTGGAGAGGCACTGTAACAGCTGAGGGTAAACAGCATAACTTTCGTAGTAGAGATCTATTGGAAGTCGTTGCGTGGATTTATAGAACTAGGAGGGAATTGCATGGACAATTCGCACGATTCCGATAGTGTATTTCTTTACCACATTCCTTGTGACAACTGTGGGAGTAGTGATGGGAACTCGCTGTTCTCTGACGGACACACGTTCTGCTACGTATGCGAGAAGTGGACTGCTGGTAATGAAGACACTAAAGAGAGGGCTTCAAAACGGAAACCCTCAGGAGGTA

AACCAATGACTTACAACGTGTGGAACTTCGGGGAATCCAATGGACGCTACTCCGCGTTAACTGCGAGAGGAATCTCCAAGGAAACCTGTCAGAAGGCTGGCTACTGGATTGCCAAAGTAGACGGTGTGATGTACCAAGTGGCTGACTATCGGGACCAGAACGGCAACATTGTGAGTCAGAAGGTTCGAGATAAAGATAAGAACTTTAAGACCACTGGTAGTCACAAGAGTGACGCTCTGTTCGGGAAGCACTTGTGGAATGGTGGTAAGAAGATTGTCGTTACAGAAGGTGAAATCGAC

ATGCTTACCGTGATGGAACTTCAAGACTGTAAGTATCCTGTAGTGTCGTTGGGTCACGGTGCCTCTGCCGCTAAGAAGACATGCGCTGCCAACTACGAATACTTTGACCAGTTCGAACAGATTATCTTAATGTTCGATATGGACGAAGCAGGGCGCAAAGCAGTCGAAGAGGCTGCACAGGTTCTACCTGCTGGTAAGGTACGAGTGGCAGTTCTTCCGTGTAAGGATGCAAACGAGTGTCACCTAAATGGTCACGACCGTGAAATCATGGAGCAAGTGTGGAATGCTGGTCCTTGGATT

CCTGATGGTGTGGTATCGGCTCTTTCGTTACGTGAACGAATCCGTGAGCACCTATCGTCCGAGGAATCAGTAGGTTTACTTTTCAGTGGCTGCACTGGTATCAACGATAAGACCTTAGGTGCCCGTGGTGGTGAAGTCATTATGGTCACTTCCGGTTCCGGTATGGGTAAGTCAACGTTCGTCCGTCAACAAGCTCTACAATGGGGCACAGCGATGGGCAAGAAGGTAGGCTTAGCGATGCTTGAGGAGTCCGTTGAGGAGACCGCTGAGGACCTTATAGGTCTACACAACCGTGTCCG

ACTGAGACAATCCGACTCACTAAAGAGAGAGATTATTGAGAACGGTAAGTTCGACCAATGGTTCGATGAACTGTTCGGCAACGATACGTTCCATCTATATGACTCATTCGCCGAGGCTGAGACGGATAGACTGCTCGCTAAGCTGGCCTACATGCGCTCAGGCTTGGGCTGTGACGTAATCATTCTAGACCACATCTCAATCGTCGTATCCGCTTCTGGTGAATCCGATGAGCGTAAGATGATTGACAACCTGATGACCAAGCTCAAAGGGTTCGCTAAGTCAACTGGGGTGGTGCTGGTC

GTAATTTGTCACCTTAAGAACCCAGACAAAGGTAAAGCACATGAGGAAGGTCGCCCCGTTTCTATTACTGACCTACGTGGTTCTGGCGCACTACGCCAACTATCTGATACTATTATTGCCCTTGAGCGTAATCAGCAAGGCGATATGCCTAACCTTGTCCTCGTTCGTATTCTCAAGTGCCGCTTTACTGGTGATACTGGTATCGCTGGCTACATGGAATACAACAAGGAAACCGGATGGCTTGAACCATCAAGTTACTCAGGGGAAGAAGAGTCACACTCAGAGTCAACAGACTGGTCCAAC

GACACTGACTTCTGACAGGATTCTTGATGACTTTCCAGACGACTACGAGAAGTTTCGCTGGAGAGTCCCATTCTAATACGACTCACTAAAGGAGACACACCATGTTCAAACTGATTAAGAAGTTAGGCCAACTGCTGGTTCGTATGTACAACGTGGAAGCCAAGCGACTGAACGATGAGGCTCGTAAAGAGGCCACACAGTCACGCGCTCTGGCGATTCGCTCCAACGAACTGGCTGACAGTGCATCCACTAAAGTTACCGAGGCTGCCCGTGTGGCAAACCAAGCTCAACAGCTTTCC

AAATTCTTTGAGTAATCAAACAGGAGAAACCATTATGTCTAACGTAGCTGAAACTATCCGTCTATCCGATACAGCTGACCAGTGGAACCGTCGAGTCCACATCAACGTTCGCAACGGTAAGGCGACTATGGTTTACCGCTGGAAGGACTCTAAGTCCTCTAAGAATCACACTCAGCGTATGACGTTGACAGATGAGCAAGCACTGCGTCTGGTCAATGCGCTTACCAAAGCTGCCGTGACAGCAATTCATGAAGCTGGTCGCGTCAATGAAGCTATGGCTATCCTCGACAAGATTGATAACT

AAGAGTGGTATCCTCAAGGTCGCCAAAGTGGTGGCCTTCATGAATACTATTCGACTCACTATAGGAGATATTACCATGCGTGACCCTAAAGTTATCCAAGCAGAAATCGCTAAACTGGAAGCTGAACTGGAGGACGTTAAGTACCATGAAGCTAAGACTCGCTCCGCTGTTCACATCTTGAAGAACTTAGGCTGGACTTGGACAAGACAGACTGGCTGGAAGAAACCAGAAGTTACCAAGCTGAGTCATAAGGTGTTCGATAAGGACACTATGACCCACATCAAGGCTGGTGATTGGGTTAA

GGTTGACATGGGAGTTGTTGGTGGATACGGCTACGTCCGCTCAGTTAGTGGCAAATATGCACAAGTGTCATACATCACAGGTGTTACTCCACGCGGTGCAATCGTTGCCGATAAGACCAACATGATTCACACAGGTTTCTTGACAGTTGTTTCATATGAAGAGATTGTTAAGTCACGATAATCAATAGGAGAAATCAATATGATCGTTTCTGACATCGAAGCTAACGCCCTCTTAGAGAGCGTCACTAAGTTCCACTGCGGGGTTATCTACGACTACTCCACCGCTGAGTACGTAAGCTACCGT

CCGAGTGACTTCGGTGCGTATCTGGATGCGCTGGAAGCCGAGGTTGCACGAGGCGGTCTTATTGTGTTCCACAACGGTCACAAGTATGACGTTCCTGCATTGACCAAACTGGCAAAGTTGCAATTGAACCGAGAGTTCCACCTTCCTCGTGAGAACTGTATTGACACCCTTGTGTTGTCACGTTTGATTCATTCCAACCTCAAGGACACCGATATGGGTCTTCTGCGTTCCGGCAAGTTGCCCGGAAAACGCTTTGGGTCTCACGCTTTGGAGGCGTGGGGTTATCGCTTAGGCGAGAT

GAAGGGTGAATACAAAGACGACTTTAAGCGTATGCTTGAAGAGCAGGGTGAAGAATACGTTGACGGAATGGAGTGGTGGAACTTCAACGAAGAGATGATGGACTATAACGTTCAGGACGTTGTGGTAACTAAAGCTCTCCTTGAGAAGCTACTCTCTGACAAACATTACTTCCCTCCTGAGATTGACTTTACGGACGTAGGATACACTACGTTCTGGTCAGAATCCCTTGAGGCCGTTGACATTGAACATCGTGCTGCATGGCTGCTCGCTAAACAAGAGCGCAACGGGTTCCCGTTTGAC

ACAAAAGCAATCGAAGAGTTGTACGTAGAGTTAGCTGCTCGCCGCTCTGAGTTGCTCCGTAAATTGACCGAAACGTTCGGCTCGTGGTATCAGCCTAAAGGTGGCACTGAGATGTTCTGCCATCCGCGAACAGGTAAGCCACTACCTAAATACCCTCGCATTAAGACACCTAAAGTTGGTGGTATCTTTAAGAAGCCTAAGAACAAGGCACAGCGAGAAGGCCGTGAGCCTTGCGAACTTGATACCCGCGAGTACGTTGCTGGTGCTCCTTACACCCCAGTTGAACATGTTGTGTTTAAC

CCTTCGTCTCGTGACCACATTCAGAAGAAACTCCAAGAGGCTGGGTGGGTCCCGACCAAGTACACCGATAAGGGTGCTCCTGTGGTGGACGATGAGGTACTCGAAGGAGTACGTGTAGATGACCCTGAGAAGCAAGCCGCTATCGACCTCATTAAAGAGTACTTGATGATTCAGAAGCGAATCGGACAGTCTGCTGAGGGAGACAAAGCATGGCTTCGTTATGTTGCTGAGGATGGTAAGATTCATGGTTCTGTTAACCCTAATGGAGCAGTTACGGGTCGTGCGACCCATGCGTTCCC

AAACCTTGCGCAAATTCCGGGTGTACGTTCTCCTTATGGAGAGCAGTGTCGCGCTGCTTTTGGCGCTGAGCACCATTTGGATGGGATAACTGGTAAGCCTTGGGTTCAGGCTGGCATCGACGCATCCGGTCTTGAGCTACGCTGCTTGGCTCACTTCATGGCTCGCTTTGATAACGGCGAGTACGCTCACGAGATTCTTAACGGCGACATCCACACTAAGAACCAGATAGCTGCTGAACTACCTACCCGAGATAACGCTAAGACGTTCATCTATGGGTTCCTCTATGGTGCTGGTGATGA

GAAGATTGGACAGATTGTTGGTGCTGGTAAAGAGCGCGGTAAGGAACTCAAGAAGAAATTCCTTGAGAACACCCCCGCGATTGCAGCACTCCGCGAGTCTATCCAACAGACACTTGTCGAGTCCTCTCAATGGGTAGCTGGTGAGCAACAAGTCAAGTGGAAACGCCGCTGGATTAAAGGTCTGGATGGTCGTAAGGTACACGTTCGTAGTCCTCACGCTGCCTTGAATACCCTACTGCAATCTGCTGGTGCTCTCATCTGCAAACTGTGGATTATCAAGACCGAAGAGATGCTCGTAG

AGAAAGGCTTGAAGCATGGCTGGGATGGGGACTTTGCGTACATGGCATGGGTACATGATGAAATCCAAGTAGGCTGCCGTACCGAAGAGATTGCTCAGGTGGTCATTGAGACCGCACAAGAAGCGATGCGCTGGGTTGGAGACCACTGGAACTTCCGGTGTCTTCTGGATACCGAAGGTAAGATGGGTCCTAATTGGGCGATTTGCCACTGATACAGGAGGCTACTCATGAACGAAAGACACTTAACAGGTGCTGCTTCTGAAATGCTAGTAGCCTACAAATTTACCAAAGCTGGGTAC

ACTGTCTATTACCCTATGCTGACTCAGAGTAAAGAGGACTTGGTTGTATGTAAGGATGGTAAATTTAGTAAGGTTCAGGTTAAAACAGCCACAACGGTTCAAACCAACACAGGAGATGCCAAGCAGGTTAGGCTAGGTGGATGCGGTAGGTCCGAATATAAGGATGGAGACTTTGACATTCTTGCGGTTGTGGTTGACGAAGATGTGCTTATTTTCACATGGGACGAAGTAAAAGGTAAGACATCCATGTGTGTCGGCAAGAGAAACAAAGGCATAAAACTATAGGAGAAATTATTATGGCTAT

GACAAAGAAATTTAAAGTGTCCTTCGACGTTACCGCAAAGATGTCGTCTGACGTTCAGGCAATCTTAGAGAAAGATATGCTGCATCTATGTAAGCAGGTCGGCTCAGGTGCGATTGTCCCCAATGGTAAACAGAAGGAAATGATTGTCCAGTTCCTGACACACGGTATGGAAGGATTGATGACATTCGTAGTACGTACATCATTTCGTGAGGCCATTAAGGACATGCACGAAGAGTATGCAGATAAGGACTCTTTCAAACAATCTCCTGCAACAGTACGGGAGGTGTTCTGATGTCTGACTAC

CTGAAAGTGCTGCAAGCAATCAAAAGTTGCCCTAAGACTTTCCAGTCCAACTATGTACGGAACAATGCGAGCCTCGTAGCGGAGGCCGCTTCCCGTGGTCACATCTCGTGCCTGACTACTAGTGGACGTAACGGTGGCGCTTGGGAAATCACTGCTTCCGGTACTCGCTTTCTGAAACGAATGGGAGGATGTGTCTAATGTCTCGTGACCTTGTGACTATTCCACGCGATGTGTGGAACGATATACAGGGCTACATCGACTCTCTGGAACGTGAGAACGATAGCCTTAAGAATCAACTAAT

GGAAGCTGACGAATACGTAGCGGAACTAGAGGAGAAACTTAATGGCACTTCTTGACCTTAAACAATTCTATGAGTTACGTGAAGGCTGCGACGACAAGGGTATCCTTGTGATGGACGGCGACTGGCTGGTCTTCCAAGCTATGAGTGCTGCTGAGTTTGATGCCTCTTGGGAGGAAGAGATTTGGCACCGATGCTGTGACCACGCTAAGGCCCGTCAGATTCTTGAGGATTCCATTAAGTCCTACGAGACCCGTAAGAAGGCTTGGGCAGGTGCTCCAATTGTCCTTGCGTTCACCGAT

AGTGTTAACTGGCGTAAAGAACTGGTTGACCCGAACTATAAGGCTAACCGTAAGGCCGTGAAGAAACCTGTAGGGTACTTTGAGTTCCTTGATGCTCTCTTTGAGCGCGAAGAGTTCTATTGCATCCGTGAGCCTATGCTTGAGGGTGATGACGTTATGGGAGTTATTGCTTCCAATCCGTCTGCCTTCGGTGCTCGTAAGGCTGTAATCATCTCTTGCGATAAGGACTTTAAGACCATCCCTAACTGTGACTTCCTGTGGTGTACCACTGGTAACATCCTGACTCAGACCGAAGAGTCCGC

TGACTGGTGGCACCTCTTCCAGACCATCAAGGGTGACATCACTGATGGTTACTCAGGGATTGCTGGATGGGGTGATACCGCCGAGGACTTCTTGAATAACCCGTTCATAACCGAGCCTAAAACGTCTGTGCTTAAGTCCGGTAAGAACAAAGGCCAAGAGGTTACTAAATGGGTTAAACGCGACCCTGAGCCTCATGAGACGCTTTGGGACTGCATTAAGTCCATTGGCGCGAAGGCTGGTATGACCGAAGAGGATATTATCAAGCAGGGCCAAATGGCTCGAATCCTACGGTTCAACGA

GTACAACTTTATTGACAAGGAGATTTACCTGTGGAGACCGTAGCGTATATTGGTCTGGGTCTTTGTGTTCTCGGAGTGTGCCTCATTTCGTGGGGCCTTTGGGACTTAGCCAGAATAATCAAGTCGTTACACGACACTAAGTGATAAACTCAAGGTCCCTAAATTAATACGACTCACTATAGGGAGATAGGGGCCTTTACGATTATTACTTTAAGATTTAACTCTAAGAGGAATCTTTATTATGTTAACACCTATTAACCAATTACTTAAGAACCCTAACGATATTCCAGATGTACCTCGTGCAACCG

CTGAGTATCTACAGGTTCGATTCAACTATGCGTACCTCGAAGCGTCTGGTCATATAGGACTTATGCGTGCTAATGGTTGTAGTGAGGCCCACATCTTGGGTTTCATTCAGGGCCTACAGTATGCCTCTAACGTCATTGACGAGATTGAGTTACGCAAGGAACAACTAAGAGATGATGGGGAGGATTGACACTATGTGTTTCTCACCGAAAATTAAAACTCCGAAGATGGATACCAATCAGATTCGAGCCGTTGAGCCAGCGCCTCTGACCCAAGAAGTGTCAAGCGTGGAGTTCGGTGGGTC

TTCTGATGAGACGGATACCGAGGGCACCGAAGTGTCTGGACGCAAAGGCCTCAAGGTCGAACGTGATGATTCCGTAGCGAAGTCTAAAGCCAGCGGCAATGGCTCCGCTCGTATGAAATCTTCCATCCGTAAGTCCGCATTTGGAGGTAAGAAGTGATGTCTGAGTTCACATGTGTGGAGGCTAAGAGTCGCTTCCGTGCAATCCGGTGGACTGTGGAACACCTTGGGTTGCCTAAAGGATTCGAAGGACACTTTGTGGGCTACAGCCTCTACGTAGACGAAGTGATGGACATGTCTG

GTTGCCGTGAAGAGTACATTCTGGACTCTACCGGAAAACATGTAGCGTACTTCGCGTGGTGCGTAAGCTGTGACATTCACCACAAAGGAGACATTCTGGATGTAACGTCCGTTGTCATTAATCCTGAGGCAGACTCTAAGGGCTTACAGCGATTCCTAGCGAAACGCTTTAAGTACCTTGCGGAACTCCACGATTGCGATTGGGTGTCTCGTTGTAAGCATGAAGGCGAGACAATGCGTGTATACTTTAAGGAGGTATAAGTTATGGGTAAGAAAGTTAAGAAGGCCGTGAAGAAAGTCAC

CAAGTCCGTTAAGAAAGTCGTTAAGGAAGGGGCTCGTCCGGTTAAACAGGTTGCTGGCGGTCTAGCTGGTCTGGCTGGTGGTACTGGTGAAGCACAGATGGTGGAAGTACCACAAGCTGCCGCACAGATTGTTGACGTACCTGAGAAAGAGGTTTCCACTGAGGACGAAGCACAGACAGAAAGCGGACGCAAGAAAGCTCGTGCTGGCGGTAAGAAATCCTTGAGTGTAGCCCGTAGCTCCGGTGGCGGTATCAACATTTAATCAGGAGGTTATCGTGGAAGACTGCATTGAATGGA

CCGGAGGTGTCAACTCTAAGGGTTATGGTCGTAAGTGGGTTAATGGTAAACTTGTGACTCCACATAGGCACATCTATGAGGAGACATATGGTCCAGTTCCAACAGGAATTGTGGTGATGCATATCTGCGATAACCCTAGGTGCTATAACATAAAGCACCTTACGCTTGGAACTCCAAAGGATAATTCCGAGGACATGGTTACCAAAGGTAGACAGGCTAAAGGAGAGGAACTAAGCAAGAAACTTACAGAGTCAGACGTTCTCGCTATACGCTCTTCAACCTTAAGCCACCGCTCCTTAGGAG

AACTGTATGGAGTCAGTCAATCAACCATAACGCGAATACTACAGCGTAAGACATGGAGACACATTTAATGGCTGAGAAACGAACAGGACTTGCGGAGGATGGCGCAAAGTCTGTCTATGAGCGTTTAAAGAACGACCGTGCTCCCTATGAGACACGCGCTCAGAATTGCGCTCAATATACCATCCCATCATTGTTCCCTAAGGACTCCGATAACGCCTCTACAGATTATCAAACTCCGTGGCAAGCCGTGGGCGCTCGTGGTCTGAACAATCTAGCCTCTAAGCTCATGCTGGCTCTATTCC

CTATGCAGACTTGGATGCGACTTACTATATCTGAATATGAAGCAAAGCAGTTACTGAGCGACCCCGATGGACTCGCTAAGGTCGATGAGGGCCTCTCGATGGTAGAGCGTATCATCATGAACTACATTGAGTCTAACAGTTACCGCGTGACTCTCTTTGAGGCTCTCAAACAGTTAGTCGTAGCTGGTAACGTCCTGCTGTACCTACCGGAACCGGAAGGGTCAAACTATAATCCCATGAAGCTGTACCGATTGTCTTCTTATGTGGTCCAACGAGACGCATTCGGCAACGTTCTGCAAATG

GTGACTCGTGACCAGATAGCTTTTGGTGCTCTCCCTGAGGACATCCGTAAGGCTGTAGAAGGTCAAGGTGGTGAGAAGAAAGCTGATGAGACAATCGACGTGTACACTCACATCTATCTGGATGAGGACTCAGGTGAATACCTCCGATACGAAGAGGTCGAGGGTATGGAAGTCCAAGGCTCCGATGGGACTTATCCTAAAGAGGCTTGCCCATACATCCCGATTCGGATGGTCAGACTAGATGGTGAATCCTACGGTCGTTCGTACATTGAGGAATACTTAGGTGACTTACGGTCCCTT

GAAAATCTCCAAGAGGCTATCGTCAAGATGTCCATGATTAGCTCTAAGGTTATCGGCTTAGTGAATCCTGCTGGTATCACCCAGCCACGCCGACTGACCAAAGCTCAGACTGGTGACTTCGTTACTGGTCGTCCAGAAGACATCTCGTTCCTCCAACTGGAGAAGCAAGCAGACTTTACTGTAGCTAAAGCCGTAAGTGACGCTATCGAGGCTCGCCTTTCGTTTGCCTTTATGTTGAACTCTGCGGTTCAGCGTACAGGTGAACGTGTGACCGCCGAAGAGATTCGGTATGTAGCTTCTG

AACTTGAAGATACTTTAGGTGGTGTCTACTCTATCCTTTCTCAAGAATTACAATTGCCTCTGGTACGAGTGCTCTTGAAGCAACTACAAGCCACGCAACAGATTCCTGAGTTACCTAAGGAAGCCGTAGAGCCAACCATTAGTACAGGTCTGGAAGCAATTGGTCGAGGACAAGACCTTGATAAGCTGGAGCGGTGTGTCACTGCGTGGGCTGCACTGGCACCTATGCGGGACGACCCTGATATTAACCTTGCGATGATTAAGTTACGTATTGCCAACGCTATCGGTATTGACACTTCTGGTA

TTCTACTCACCGAAGAACAGAAGCAACAGAAGATGGCCCAACAGTCTATGCAAATGGGTATGGATAATGGTGCTGCTGCGCTGGCTCAAGGTATGGCTGCACAAGCTACAGCTTCACCTGAGGCTATGGCTGCTGCCGCTGATTCCGTAGGTTTACAGCCGGGAATTTAATACGACTCACTATAGGGAGACCTCATCTTTGAAATGAGCGATGACAAGAGGTTGGAGTCCTCGGTCTTCCTGTAGTTCAACTTTAAGGAGACAATAATAATGGCTGAATCTAATGCAGACGTATATGCATCTT

TTGGCGTGAACTCCGCTGTGATGTCTGGTGGTTCCGTTGAGGAACATGAGCAGAACATGCTGGCTCTTGATGTTGCTGCCCGTGATGGCGATGATGCAATCGAGTTAGCGTCAGACGAAGTGGAAACAGAACGTGACCTGTATGACAACTCTGACCCGTTCGGTCAAGAGGATGACGAAGGCCGCATTCAGGTTCGTATCGGTGATGGCTCTGAGCCGACCGATGTGGACACTGGAGAAGAAGGCGTTGAGGGCACCGAAGGTTCCGAAGAGTTTACCCCACTGGGCGAGACTCCA

GAAGAACTGGTAGCTGCCTCTGAGCAACTTGGTGAGCACGAAGAGGGCTTCCAAGAGATGATTAACATTGCTGCTGAGCGTGGCATGAGTGTCGAGACCATTGAGGCTATCCAGCGTGAGTACGAGGAGAACGAAGAGTTGTCCGCCGAGTCCTACGCTAAGCTGGCTGAAATTGGCTACACGAAGGCTTTCATTGACTCGTATATCCGTGGTCAAGAAGCTCTGGTGGAGCAGTACGTAAACAGTGTCATTGAGTACGCTGGTGGTCGTGAACGTTTTGATGCACTGTATAACCACCT

TGAGACGCACAACCCTGAGGCTGCACAGTCGCTGGATAATGCGTTGACCAATCGTGACTTAGCGACCGTTAAGGCTATCATCAACTTGGCTGGTGAGTCTCGCGCTAAGGCGTTCGGTCGTAAGCCAACTCGTAGTGTGACTAATCGTGCTATTCCGGCTAAACCTCAGGCTACCAAGCGTGAAGGCTTTGCGGACCGTAGCGAGATGATTAAAGCTATGAGTGACCCTCGGTATCGCACAGATGCCAACTATCGTCGTCAAGTCGAACAGAAAGTAATCGATTCGAACTTCTGATAGAC

TTCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGGCTAGCATGACTGGTGGACAGCAAATGGGTACTAACCAAGGTAAAGGTGTAGTTGCTGCTGGAGATAAACTGGCGTTGTTCTTGAAGGTATTTGGCGGTGAAGTCCTGACTGCGTTCGCTCGTACCTCCGTGACCACTTCTCGCCACATGGTACGTTCCATCTCCAGCGGTAAATCCGCTCAGTTCCCTGTTCTGGGTCGCACTCAGGCAGC

GTATCTGGCTCCGGGCGAGAACCTCGACGATAAACGTAAGGACATCAAACACACCGAGAAGGTAATCACCATTGACGGTCTCCTGACGGCTGACGTTCTGATTTATGATATTGAGGACGCGATGAACCACTACGACGTTCGCTCTGAGTATACCTCTCAGTTGGGTGAATCTCTGGCGATGGCTGCGGATGGTGCGGTTCTGGCTGAGATTGCCGGTCTGTGTAACGTGGAAAGCAAATATAATGAGAACATCGAGGGCTTAGGTACTGCTACCGTAATTGAGACCACTCAGAACAAGGC

CGCACTTACCGACCAAGTTGCGCTGGGTAAGGAGATTATTGCGGCTCTGACTAAGGCTCGTGCGGCTCTGACCAAGAACTATGTTCCGGCTGCTGACCGTGTGTTCTACTGTGACCCAGATAGCTACTCTGCGATTCTGGCAGCACTGATGCCGAACGCAGCAAACTACGCTGCTCTGATTGACCCTGAGAAGGGTTCTATCCGCAACGTTATGGGCTTTGAGGTTGTAGAAGTTCCGCACCTCACCGCTGGTGGTGCTGGTACCGCTCGTGAGGGCACTACTGGTCAGAAGCACGTC

TTCCCTGCCAATAAAGGTGAGGGTAATGTCAAGGTTGCTAAGGACAACGTTATCGGCCTGTTCATGCACCGCTCTGCGGTAGGTACTGTTAAGCTGCGTGACTTGGCTCTGGAGCGCGCTCGCCGTGCTAACTTCCAAGCGGACCAGATTATCGCTAAGTACGCAATGGGCCACGGTGGTCTTCGCCCAGAAGCTGCTGGTGCAGTGGTTTTCAAAGTGGAGTAATGCTGGGGGTGGCCTCAACGGTCGCTGCTAGTCCCGAAGAGGCGAGTGTTACTTCAACAGAAGAAACCTTAA

CGCCAGCACAGGAGGCCGCACGCACCCGCGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATGCGCTCATACGATATGAACGTTGAGACTGCCGCTGAGTTATCAGCTGTGAACGACATTCTGGCGTCTATCGGTGAACCTCCGGTATCAACGCTGGAAGGTGACGCTAACGCAGATGCAGCGAACGCTCGGCGTATTCTCAACAA

GATTAACCGACAGATTCAATCTCGTGGATGGACGTTCAACATTGAGGAAGGCATAACGCTACTACCTGATGTTTACTCCAACCTGATTGTATACAGTGACGACTATTTATCCCTAATGTCTACTTCCGGTCAATCCATCTACGTTAACCGAGGTGGCTATGTGTATGACCGAACGAGTCAATCAGACCGCTTTGACTCTGGTATTACTGTGAACATTATTCGTCTCCGCGACTACGATGAGATGCCTGAGTGCTTCCGTTACTGGATTGTCACCAAGGCTTCCCGTCAGTTCAACAACCGATTCT

TTGGGGCACCGGAAGTAGAGGGTGTACTCCAAGAAGAGGAAGATGAGGCTAGACGTCTCTGCATGGAGTATGAGATGGACTACGGTGGGTACAATATGCTGGATGGAGATGCGTTCACTTCTGGTCTACTGACTCGCTAACATTAATAAATAAGGAGGCTCTAATGGCACTCATTAGCCAATCAATCAAGAACTTGAAGGGTGGTATCAGCCAACAGCCTGACATCCTTCGTTATCCAGACCAAGGGTCACGCCAAGTTAACGGTTGGTCTTCGGAGACCGAGGGCCTCCAAAAGCGTCC

ACCTCTTGTTTTCTTAAATACACTTGGAGACAACGGTGCGTTAGGTCAAGCTCCGTACATCCACCTGATTAACCGAGATGAGCACGAACAGTATTACGCTGTGTTCACTGGTAGCGGAATCCGAGTGTTCGACCTTTCTGGTAACGAGAAGCAAGTTAGGTATCCTAACGGTTCCAACTACATCAAGACCGCTAATCCACGTAACGACCTGCGAATGGTTACTGTAGCAGACTATACGTTCATCGTTAACCGTAACGTTGTTGCACAGAAGAACACAAAGTCTGTCAACTTACCGAATTACAAC

CCTAATCAAGACGGATTGATTAACGTTCGTGGTGGTCAGTATGGTAGGGAACTAATTGTACACATTAACGGTAAAGACGTTGCGAAGTATAAGATACCAGATGGTAGTCAACCTGAACACGTAAACAATACGGATGCCCAATGGTTAGCTGAAGAGTTAGCCAAGCAGATGCGCACTAACTTGTCTGATTGGACTGTAAATGTAGGGCAAGGGTTCATCCATGTGACCGCACCTAGTGGTCAACAGATTGACTCCTTCACGACTAAAGATGGCTACGCAGACCAGTTGATTAACCCTGTGACC

CACTACGCTCAGTCGTTCTCTAAGCTGCCACCTAATGCTCCTAACGGCTACATGGTGAAAATCGTAGGGGACGCCTCTAAGTCTGCCGACCAGTATTACGTTCGGTATGACGCTGAGCGGAAAGTTTGGACTGAGACTTTAGGTTGGAACACTGAGGACCAAGTTCTATGGGAAACCATGCCACACGCTCTTGTGCGAGCCGCTGACGGTAATTTCGACTTCAAGTGGCTTGAGTGGTCTCCTAAGTCTTGTGGTGACGTTGACACCAACCCTTGGCCTTCTTTTGTTGGTTCAAGTATTA

ACGATGTGTTCTTCTTCCGTAACCGCTTAGGATTCCTTAGTGGGGAGAACATCATATTGAGTCGTACAGCCAAATACTTCAACTTCTACCCTGCGTCCATTGCGAACCTTAGTGATGACGACCCTATAGACGTAGCTGTGAGTACCAACCGAATAGCAATCCTTAAGTACGCCGTTCCGTTCTCAGAAGAGTTACTCATCTGGTCCGATGAAGCACAATTCGTCCTGACTGCCTCGGGTACTCTCACATCTAAGTCGGTTGAGTTGAACCTAACGACCCAGTTTGACGTACAGGACCGAGCGA

GACCTTTTGGGATTGGGCGTAATGTCTACTTTGCTAGTCCGAGGTCCAGCTTCACGTCCATCCACAGGTACTACGCTGTGCAGGATGTCAGTTCCGTTAAGAATGCTGAGGACATTACATCACACGTTCCTAACTACATCCCTAATGGTGTGTTCAGTATTTGCGGAAGTGGTACGGAAAACTTCTGTTCGGTACTATCTCACGGGGACCCTAGTAAAATCTTCATGTACAAATTCCTGTACCTGAACGAAGAGTTAAGGCAACAGTCGTGGTCTCATTGGGACTTTGGGGAAAACGTACAGG

TTCTAGCTTGTCAGAGTATCAGCTCAGATATGTATGTGATTCTTCGCAATGAGTTCAATACGTTCCTAGCTAGAATCTCTTTCACTAAGAACGCCATTGACTTACAGGGAGAACCCTATCGTGCCTTTATGGACATGAAGATTCGATACACGATTCCTAGTGGAACATACAACGATGACACATTCACTACCTCTATTCATATTCCAACAATTTATGGTGCAAACTTCGGGAGGGGCAAAATCACTGTATTGGAGCCTGATGGTAAGATAACCGTGTTTGAGCAACCTACGGCTGGGTGGAATAGCG

ACCCTTGGCTGAGACTCAGCGGTAACTTGGAGGGACGCATGGTGTACATTGGGTTCAACATTAACTTCGTATATGAGTTCTCTAAGTTCCTCATCAAGCAGACTGCCGACGACGGGTCTACCTCCACGGAAGACATTGGGCGCTTACAGTTACGCCGAGCGTGGGTTAACTACGAGAACTCTGGTACGTTTGACATTTATGTTGAGAACCAATCGTCTAACTGGAAGTACACAATGGCTGGTGCCCGATTAGGCTCTAACACTCTGAGGGCTGGGAGACTGAACTTAGGGACCGGACAAT

ATCGATTCCCTGTGGTTGGTAACGCCAAGTTCAACACTGTATACATCTTGTCAGATGAGACTACCCCTCTGAACATCATTGGGTGTGGCTGGGAAGGTAACTACTTACGGAGAAGTTCCGGTATTTAATTAAATATTCTCCCTGTGGTGGCTCGAAATTAATACGACTCACTATAGGGAGAACAATACGACTACGGGAGGGTTTTCTTATGATGACTATAAGACCTACTAAAAGTACAGACTTTGAGGTATTCACTCCGGCTCACCATGACATTCTTGAAGCTAAGGCTGCTGGTATTGAGCCGA

GTTTCCCTGATGCTTCCGAGTGTGTCACGTTGAGCCTCTATGGGTTCCCTCTAGCTATCGGTGGTAACTGCGGGGACCAGTGCTGGTTCGTTACGAGCGACCAAGTGTGGCGACTTAGTGGAAAGGCTAAGCGAAAGTTCCGTAAGTTAATCATGGAGTATCGCGATAAGATGCTTGAGAAGTATGATACTCTTTGGAATTACGTATGGGTAGGCAATACGTCCCACATTCGTTTCCTCAAGACTATCGGTGCGGTATTCCATGAAGAGTACACACGAGATGGTCAATTTCAGTTATTTACAA

TCACGAAAGGAGGATAACCATATGTGTTGGGCAGCCGCAATACCTATCGCTATATCTGGCGCTCAGGCTATCAGTGGTCAGAACGCTCAGGCCAAAATGATTGCCGCTCAGACCGCTGCTGGTCGTCGTCAAGCTATGGAAATCATGAGGCAGACGAACATCCAGAATGCTGACCTATCGTTGCAAGCTCGAAGTAAACTTGAGGAAGCGTCCGCCGAGTTGACCTCACAGAACATGCAGAAGGTCCAAGCTATTGGGTCTATCCGAGCGGCTATCGGAGAGAGTATGCTTGAAGGTTC

CTCAATGGACCGCATTAAGCGAGTCACAGAAGGACAGTTCATTCGGGAAGCCAATATGGTAACTGAGAACTATCGCCGTGACTACCAAGCAATCTTCGCACAGCAACTTGGTGGTACTCAAAGTGCTGCAAGTCAGATTGACGAAATCTATAAGAGCGAACAGAAACAGAAGAGTAAGCTACAGATGGTTCTGGACCCACTGGCTATCATGGGGTCTTCCGCTGCGAGTGCTTACGCATCCGGTGCGTTCGACTCTAAGTCCACAACTAAGGCACCTATTGTTGCCGCTAAAGGAACCAA

GACGGGGAGGTAATGAGCTATGAGTAAAATTGAATCTGCCCTTCAAGCGGCACAACCGGGACTCTCTCGGTTACGTGGTGGTGCTGGAGGTATGGGCTATCGTGCAGCAACCACTCAGGCCGAACAGCCAAGGTCAAGCCTATTGGACACCATTGGTCGGTTCGCTAAGGCTGGTGCCGATATGTATACCGCTAAGGAACAACGAGCACGAGACCTAGCTGATGAACGCTCTAACGAGATTATCCGTAAGCTGACCCCTGAGCAACGTCGAGAAGCTCTCAACAACGGGACCCTTCTG

TATCAGGATGACCCATACGCTATGGAAGCACTCCGAGTCAAGACTGGTCGTAACGCTGCGTATCTTGTGGACGATGACGTTATGCAGAAGATAAAAGAGGGTGTCTTCCGTACTCGCGAAGAGATGGAAGAGTATCGCCATAGTCGCCTTCAAGAGGGCGCTAAGGTATACGCTGAGCAGTTCGGCATCGACCCTGAGGACGTTGATTATCAGCGTGGTTTCAACGGGGACATTACCGAGCGTAACATCTCGCTGTATGGTGCGCATGATAACTTCTTGAGCCAGCAAGCTCAGAAGGGC

GCTATCATGAACAGCCGAGTGGAACTCAACGGTGTCCTTCAAGACCCTGATATGCTGCGTCGTCCAGACTCTGCTGACTTCTTTGAGAAGTATATCGACAACGGTCTGGTTACTGGCGCAATCCCATCTGATGCTCAAGCCACACAGCTTATAAGCCAAGCGTTCAGTGACGCTTCTAGCCGTGCTGGTGGTGCTGACTTCCTGATGCGAGTCGGTGACAAGAAGGTAACACTTAACGGAGCCACTACGACTTACCGAGAGTTGATTGGTGAGGAACAGTGGAACGCTCTCATGGTCAC

AGCACAACGTTCTCAGTTTGAGACTGACGCGAAGCTGAACGAGCAGTATCGCTTGAAGATTAACTCTGCGCTGAACCAAGAGGACCCAAGGACAGCTTGGGAGATGCTTCAAGGTATCAAGGCTGAACTAGATAAGGTCCAACCTGATGAGCAGATGACACCACAACGTGAGTGGCTAATCTCCGCACAGGAACAAGTTCAGAATCAGATGAACGCATGGACGAAAGCTCAGGCCAAGGCTCTGGACGATTCCATGAAGTCAATGAACAAACTTGACGTAATCGACAAGCAATTCCAGA

AGCGAATCAACGGTGAGTGGGTCTCAACGGATTTTAAGGATATGCCAGTCAACGAGAACACTGGTGAGTTCAAGCATAGCGATATGGTTAACTACGCCAATAAGAAGCTCGCTGAGATTGACAGTATGGACATTCCAGACGGTGCCAAGGATGCTATGAAGTTGAAGTACCTTCAAGCGGACTCTAAGGACGGAGCATTCCGTACAGCCATCGGAACCATGGTCACTGACGCTGGTCAAGAGTGGTCTGCCGCTGTGATTAACGGTAAGTTACCAGAACGAACCCCAGCTATGGATGCTC

TGCGCAGAATCCGCAATGCTGACCCTCAGTTGATTGCTGCGCTATACCCAGACCAAGCTGAGCTATTCCTGACGATGGACATGATGGACAAGCAGGGTATTGACCCTCAGGTTATTCTTGATGCCGACCGACTGACTGTTAAGCGGTCCAAAGAGCAACGCTTTGAGGATGATAAAGCATTCGAGTCTGCACTGAATGCATCTAAGGCTCCTGAGATTGCCCGTATGCCAGCGTCACTGCGCGAATCTGCACGTAAGATTTATGACTCCGTTAAGTATCGCTCGGGGAACGAAAGCATGG

CTATGGAGCAGATGACCAAGTTCCTTAAGGAATCTACCTACACGTTCACTGGTGATGATGTTGACGGTGATACCGTTGGTGTGATTCCTAAGAATATGATGCAGGTTAACTCTGACCCGAAATCATGGGAGCAAGGTCGGGATATTCTGGAGGAAGCACGTAAGGGAATCATTGCGAGCAACCCTTGGATAACCAATAAGCAACTGACCATGTATTCTCAAGGTGACTCCATTTACCTTATGGACACCACAGGTCAAGTCAGAGTCCGATACGACAAAGAGTTACTCTCGAAGGTCTGGAGT

GAGAACCAGAAGAAACTCGAAGAGAAAGCTCGTGAGAAGGCTCTGGCTGATGTGAACAAGCGAGCACCTATAGTTGCCGCTACGAAGGCCCGTGAAGCTGCTGCTAAACGAGTCCGAGAGAAACGTAAACAGACTCCTAAGTTCATCTACGGACGTAAGGAGTAACTAAAGGCTACATAAGGAGGCCCTAAATGGATAAGTACGATAAGAACGTACCAAGTGATTATGATGGTCTGTTCCAAAAGGCTGCTGATGCCAACGGGGTCTCTTATGACCTTTTACGTAAAGTCGCTTGGACAG

AATCACGATTTGTGCCTACAGCAAAATCTAAGACTGGACCATTAGGCATGATGCAATTTACCAAGGCAACCGCTAAGGCCCTCGGTCTGCGAGTTACCGATGGTCCAGACGACGACCGACTGAACCCTGAGTTAGCTATTAATGCTGCCGCTAAGCAACTTGCAGGTCTGGTAGGGAAGTTTGATGGCGATGAACTCAAAGCTGCCCTTGCGTACAACCAAGGCGAGGGACGCTTGGGTAATCCACAACTTGAGGCGTACTCTAAGGGAGACTTCGCATCAATCTCTGAGGAGGGACGT

AACTACATGCGTAACCTTCTGGATGTTGCTAAGTCACCTATGGCTGGACAGTTGGAAACTTTTGGTGGCATAACCCCAAAGGGTAAAGGCATTCCGGCTGAGGTAGGATTGGCTGGAATTGGTCACAAGCAGAAAGTAACACAGGAACTTCCTGAGTCCACAAGTTTTGACGTTAAGGGTATCGAACAGGAGGCTACGGCGAAACCATTCGCCAAGGACTTTTGGGAGACCCACGGAGAAACACTTGACGAGTACAACAGTCGTTCAACCTTCTTCGGATTCAAAAATGCTGCCGAAGC

TGAACTCTCCAACTCAGTCGCTGGGATGGCTTTCCGTGCTGGTCGTCTCGATAATGGTTTTGATGTGTTTAAAGACACCATTACGCCGACTCGCTGGAACTCTCACATCTGGACTCCAGAGGAGTTAGAGAAGATTCGAACAGAGGTTAAGAACCCTGCGTACATCAACGTTGTAACTGGTGGTTCCCCTGAGAACCTCGATGACCTCATTAAATTGGCTAACGAGAACTTTGAGAATGACTCCCGCGCTGCCGAGGCTGGCCTAGGTGCCAAACTGAGTGCTGGTATTATTGGTGCTGG

TGTGGACCCGCTTAGCTATGTTCCTATGGTCGGTGTCACTGGTAAGGGCTTTAAGTTAATCAATAAGGCTCTTGTAGTTGGTGCCGAAAGTGCTGCTCTGAACGTTGCATCCGAAGGTCTCCGTACCTCCGTAGCTGGTGGTGACGCAGACTATGCGGGTGCTGCCTTAGGTGGCTTTGTGTTTGGCGCAGGCATGTCTGCAATCAGTGACGCTGTAGCTGCTGGACTGAAACGCAGTAAACCAGAAGCTGAGTTCGACAATGAGTTCATCGGTCCTATGATGCGATTGGAAGCCCGTG

AGACAGCACGAAACGCCAACTCTGCGGACCTCTCTCGGATGAACACTGAGAACATGAAGTTTGAAGGTGAACATAATGGTGTCCCTTATGAGGACTTACCAACAGAGAGAGGTGCCGTGGTGTTACATGATGGCTCCGTTCTAAGTGCAAGCAACCCAATCAACCCTAAGACTCTAAAAGAGTTCTCCGAGGTTGACCCTGAGAAGGCTGCGCGAGGAATCAAACTGGCTGGGTTCACCGAGATTGGCTTGAAGACCTTGGGGTCTGACGATGCTGACATCCGTAGAGTGGCTATCGA

CCTCGTTCGCTCTCCTACTGGTATGCAGTCTGGTGCCTCAGGTAAGTTCGGTGCAACAGCTTCTGACATCCATGAGAGACTTCATGGTACTGACCAGCGTACTTATAATGACTTGTACAAAGCAATGTCTGACGCTATGAAAGACCCTGAGTTCTCTACTGGCGGCGCTAAGATGTCCCGTGAAGAAACTCGATACACTATCTACCGTAGAGCGGCACTAGCTATTGAGCGTCCAGAACTACAGAAGGCACTCACTCCGTCTGAGAGAATCGTTATGGACATCATTAAGCGTCACTTTGACA

CCAAGCGTGAACTTATGGAAAACCCAGCAATATTCGGTAACACAAAGGCTGTGAGTATCTTCCCTGAGAGTCGCCACAAAGGTACTTACGTTCCTCACGTATATGACCGTCATGCCAAGGCGCTGATGATTCAACGCTACGGTGCCGAAGGTTTGCAGGAAGGGATTGCCCGCTCATGGATGAACAGCTACGTCTCCAGACCTGAGGTCAAGGCCAGAGTCGATGAGATGCTTAAGGAATTACACGGGGTGAAGGAAGTAACACCAGAGATGGTAGAGAAGTACGCTATGGATAAGGCT

TATGGTATCTCCCACTCAGACCAGTTCACCAACAGTTCCATAATAGAAGAGAACATTGAGGGCTTAGTAGGTATCGAGAATAACTCATTCCTTGAGGCACGTAACTTGTTTGATTCGGACCTATCCATCACTATGCCAGACGGACAGCAATTCTCAGTGAATGACCTAAGGGACTTCGATATGTTCCGCATCATGCCAGCGTATGACCGCCGTGTCAATGGTGACATCGCCATCATGGGGTCTACTGGTAAAACCACTAAGGAACTTAAGGATGAGATTTTGGCTCTCAAAGCGAAAGCTGAG

GGAGACGGTAAGAAGACTGGCGAGGTACATGCTTTAATGGATACCGTTAAGATTCTTACTGGTCGTGCTAGACGCAATCAGGACACTGTGTGGGAAACCTCACTGCGTGCCATCAATGACCTAGGGTTCTTCGCTAAGAACGCCTACATGGGTGCTCAGAACATTACGGAGATTGCTGGGATGATTGTCACTGGTAACGTTCGTGCTCTAGGGCATGGTATCCCAATTCTGCGTGATACACTCTACAAGTCTAAACCAGTTTCAGCTAAGGAACTCAAGGAACTCCATGCGTCTCTGTTCG

GGAAGGAGGTGGACCAGTTGATTCGGCCTAAACGTGCTGACATTGTGCAGCGCCTAAGGGAAGCAACTGATACCGGACCTGCCGTGGCGAACATCGTAGGGACCTTGAAGTATTCAACACAGGAACTGGCTGCTCGCTCTCCGTGGACTAAGCTACTGAACGGAACCACTAACTACCTTCTGGATGCTGCGCGTCAAGGTATGCTTGGGGATGTTATTAGTGCCACCCTAACAGGTAAGACTACCCGCTGGGAGAAAGAAGGCTTCCTTCGTGGTGCCTCCGTAACTCCTGAGCAGAT

GGCTGGCATCAAGTCTCTCATCAAGGAACATATGGTACGCGGTGAGGACGGGAAGTTTACCGTTAAGGACAAGCAAGCGTTCTCTATGGACCCACGGGCTATGGACTTATGGAGACTGGCTGACAAGGTAGCTGATGAGGCAATGCTGCGTCCACATAAGGTGTCCTTACAGGATTCCCATGCGTTCGGAGCACTAGGTAAGATGGTTATGCAGTTTAAGTCTTTCACTATCAAGTCCCTTAACTCTAAGTTCCTGCGAACCTTCTATGATGGATACAAGAACAACCGAGCGATTGACGCT

GCGCTGAGCATCATCACCTCTATGGGTCTCGCTGGTGGTTTCTATGCTATGGCTGCACACGTCAAAGCATACGCTCTGCCTAAGGAGAAACGTAAGGAGTACTTGGAGCGTGCACTGGACCCAACCATGATTGCCCACGCTGCGTTATCTCGTAGTTCTCAATTGGGTGCTCCTTTGGCTATGGTTGACCTAGTTGGTGGTGTTTTAGGGTTCGAGTCCTCCAAGATGGCTCGCTCTACGATTCTACCTAAGGACACCGTGAAGGAACGTGACCCAAACAAACCGTACACCTCTAGAGAG

GTAATGGGCGCTATGGGTTCAAACCTTCTGGAACAGATGCCTTCGGCTGGCTTTGTGGCTAACGTAGGGGCTACCTTAATGAATGCTGCTGGCGTGGTCAACTCACCTAATAAAGCAACCGAGCAGGACTTCATGACTGGTCTTATGAACTCCACAAAAGAGTTAGTACCGAACGACCCATTGACTCAACAGCTTGTGTTGAAGATTTATGAGGCGAACGGTGTTAACTTGAGGGAGCGTAGGAAATAATACGACTCACTATAGGGAGAGGCGAAATAATCTTCTCCCTGTAGTCTCTTAGA

TTTACTTTAAGGAGGTCAAATGGCTAACGTAATTAAAACCGTTTTGACTTACCAGTTAGATGGCTCCAATCGTGATTTTAATATCCCGTTTGAGTATCTAGCCCGTAAGTTCGTAGTGGTAACTCTTATTGGTGTAGACCGAAAGGTCCTTACGATTAATACAGACTATCGCTTTGCTACACGTACTACTATCTCTCTGACAAAGGCTTGGGGTCCAGCCGATGGCTACACGACCATCGAGTTACGTCGAGTAACCTCCACTACCGACCGATTGGTTGACTTTACGGATGGTTCAATCCTCCGCGC

GTATGACCTTAACGTCGCTCAGATTCAAACGATGCACGTAGCGGAAGAGGCCCGTGACCTCACTACGGATACTATCGGTGTCAATAACGATGGTCACTTGGATGCTCGTGGTCGTCGAATTGTGAACCTAGCGAACGCCGTGGATGACCGCGATGCTGTTCCGTTTGGTCAACTAAAGACCATGAACCAGAACTCATGGCAAGCACGTAATGAAGCCTTACAGTTCCGTAATGAGGCTGAGACTTTCAGAAACCAAGCGGAGGGCTTTAAGAACGAGTCCAGTACCAACGCTACGAACA

CAAAGCAGTGGCGCGATGAGACCAAGGGTTTCCGAGACGAAGCCAAGCGGTTCAAGAATACGGCTGGTCAATACGCTACATCTGCTGGGAACTCTGCTTCCGCTGCGCATCAATCTGAGGTAAACGCTGAGAACTCTGCCACAGCATCCGCTAACTCTGCTCATTTGGCAGAACAGCAAGCAGACCGTGCGGAACGTGAGGCAGACAAGCTGGAAAATTACAATGGATTGGCTGGTGCAATTGATAAGGTAGATGGAACCAATGTGTACTGGAAAGGAAATATTCACGCTAACGGGCG

CCTTTACATGACCACAAACGGTTTTGACTGTGGCCAGTATCAACAGTTCTTTGGTGGTGTCACTAATCGTTACTCTGTCATGGAGTGGGGAGATGAGAACGGATGGCTGATGTATGTTCAACGTAGAGAGTGGACAACAGCGATAGGCGGTAACATCCAGTTAGTAGTAAACGGACAGATCATCACCCAAGGTGGAGCCATGACCGGTCAGCTAAAATTGCAGAATGGGCATGTTCTTCAATTAGAGTCCGCATCCGACAAGGCGCACTATATTCTATCTAAAGATGGTAACAGGAATAACT

GGTACATTGGTAGAGGGTCAGATAACAACAATGACTGTACCTTCCACTCCTATGTACATGGTACGACCTTAACACTCAAGCAGGACTATGCAGTAGTTAACAAACACTTCCACGTAGGTCAGGCCGTTGTGGCCACTGATGGTAATATTCAAGGTACTAAGTGGGGAGGTAAATGGCTGGATGCTTACCTACGTGACAGCTTCGTTGCGAAGTCCAAGGCGTGGACTCAGGTGTGGTCTGGTAGTGCTGGCGGTGGGGTAAGTGTGACTGTTTCACAGGATCTCCGCTTCCGCAATATCTG

GATTAAGTGTGCCAACAACTCTTGGAACTTCTTCCGTACTGGCCCCGATGGAATCTACTTCATAGCCTCTGATGGTGGATGGTTACGATTCCAAATACACTCCAACGGTCTCGGATTCAAGAATATTGCAGACAGTCGTTCAGTACCTAATGCAATCATGGTGGAGAACGAGTAATTGGTAAATCACAAGGAAAGACGTGTAGTCCACGGATGGACTCTCAAGGAGGTACAAGGTGCTATCATTAGACTTTAACAACGAATTGATTAAGGCTGCTCCAATTGTTGGGACGGGTGTAGCAGATG

TTAGTGCTCGACTGTTCTTTGGGTTAAGCCTTAACGAATGGTTCTACGTTGCTGCTATCGCCTACACAGTGGTTCAGATTGGTGCCAAGGTAGTCGATAAGATGATTGACTGGAAGAAAGCCAATAAGGAGTGATATGTATGGAAAAGGATAAGAGCCTTATTACATTCTTAGAGATGTTGGACACTGCGATGGCTCAGCGTATGCTTGCGGACCTTTCGGACCATGAGCGTCGCTCTCCGCAACTCTATAATGCTATTAACAAACTGTTAGACCGCCACAAGTTCCAGATTGGTAAGTTGCAG

CCGGATGTTCACATCTTAGGTGGCCTTGCTGGTGCTCTTGAAGAGTACAAAGAGAAAGTCGGTGATAACGGTCTTACGGATGATGATATTTACACATTACAGTGATATACTCAAGGCCACTACAGATAGTGGTCTTTATGGATGTCATTGTCTATACGAGATGCTCCTACGTGAAATCTGAAAGTTAACGGGAGGCATTATGCTAGAATTTTTACGTAAGCTAATCCCTTGGGTTCTCGCTGGGATGCTATTCGGGTTAGGATGGCATCTAGGGTCAGACTCAATGGACGCTAAATGGAAACAGG

AGGTACACAATGAGTACGTTAAGAGAGTTGAGGCTGCGAAGAGCACTCAAAGAGCAATCGATGCGGTATCTGCTAAGTATCAAGAAGACCTTGCCGCGCTGGAAGGGAGCACTGATAGGATTATTTCTGATTTGCGTAGCGACAATAAGCGGTTGCGCGTCAGAGTCAAAACTACCGGAACCTCCGATGGTCAGTGTGGATTCGAGCCTGATGGTCGAGCCGAACTTGACGACCGAGATGCTAAACGTATTCTCGCAGTGACCCAGAAGGGTGACGCATGGATTCGTGCGTTACAGGAT

ACTATTCGTGAACTGCAACGTAAGTAGGAAATCAAGTAAGGAGGCAATGTGTCTACTCAATCCAATCGTAATGCGCTCGTAGTGGCGCAACTGAAAGGAGACTTCGTGGCGTTCCTATTCGTCTTATGGAAGGCGCTAAACCTACCGGTGCCCACTAAGTGTCAGATTGACATGGCTAAGGTGCTGGCGAATGGAGACAACAAGAAGTTCATCTTACAGGCTTTCCGTGGTATCGGTAAGTCGTTCATCACATGTGCGTTCGTTGTGTGGTCCTTATGGAGAGACCCTCAGTTGAAGATACT

TATCGTATCAGCCTCTAAGGAGCGTGCAGACGCTAACTCCATCTTTATTAAGAACATCATTGACCTGCTGCCATTCCTATCTGAGTTAAAGCCAAGACCCGGACAGCGTGACTCGGTAATCAGCTTTGATGTAGGCCCAGCCAATCCTGACCACTCTCCTAGTGTGAAATCAGTAGGTATCACTGGTCAGTTAACTGGTAGCCGTGCTGACATTATCATTGCGGATGACGTTGAGATTCCGTCTAACAGCGCAACTATGGGTGCCCGTGAGAAGCTATGGACTCTGGTTCAGGAGTTCGCTG

CGTTACTTAAACCGCTGCCTTCCTCTCGCGTTATCTACCTTGGTACACCTCAGACAGAGATGACTCTCTATAAGGAACTTGAGGATAACCGTGGGTACACAACCATTATCTGGCCTGCTCTGTACCCAAGGACACGTGAAGAGAACCTCTATTACTCACAGCGTCTTGCTCCTATGTTACGCGCTGAGTACGATGAGAACCCTGAGGCACTTGCTGGGACTCCAACAGACCCAGTGCGCTTTGACCGTGATGACCTGCGCGAGCGTGAGTTGGAATACGGTAAGGCTGGCTTTACGCTAC

AGTTCATGCTTAACCCTAACCTTAGTGATGCCGAGAAGTACCCGCTGAGGCTTCGTGACGCTATCGTAGCGGCCTTAGACTTAGAGAAGGCCCCAATGCATTACCAGTGGCTTCCGAACCGTCAGAACATCATTGAGGACCTTCCTAACGTTGGCCTTAAGGGTGATGACCTGCATACGTACCACGATTGTTCCAACAACTCAGGTCAGTACCAACAGAAGATTCTGGTCATTGACCCTAGTGGTCGCGGTAAGGACGAAACAGGTTACGCTGTGCTGTACACACTGAACGGTTACATCTA

CCTTATGGAAGCTGGAGGTTTCCGTGATGGCTACTCCGATAAGACCCTTGAGTTACTCGCTAAGAAGGCAAAGCAATGGGGAGTCCAGACGGTTGTCTACGAGAGTAACTTCGGTGACGGTATGTTCGGTAAGGTATTCAGTCCTATCCTTCTTAAACACCACAACTGTGCGATGGAAGAGATTCGTGCCCGTGGTATGAAAGAGATGCGTATTTGCGATACCCTTGAGCCAGTCATGCAGACTCACCGCCTTGTAATTCGTGATGAGGTCATTAGGGCCGACTACCAGTCCGCTCGTGAC

GTAGACGGTAAGCATGACGTTAAGTACTCGTTGTTCTACCAGATGACCCGTATCACTCGTGAGAAAGGCGCTCTGGCTCATGATGACCGATTGGATGCCCTTGCGTTAGGCATTGAGTATCTCCGTGAGTCCATGCAGTTGGATTCCGTTAAGGTCGAGGGTGAAGTACTTGCTGACTTCCTTGAGGAACACATGATGCGTCCTACGGTTGCTGCTACGCATATCATTGAGATGTCTGTGGGAGGAGTTGATGTGTACTCTGAGGACGATGAGGGTTACGGTACGTCTTTCATTGAGTGGT

GATTTATGCATTAGGACTGCATAGGGATGCACTATAGACCACGGATGGTCAGTTCTTTAAGTTACTGAAAAGACACGATAAATTAATACGACTCACTATAGGGAGAGGAGGGACGAAAGGTTACTATATAGATACTGAATGAATACTTATAGAGTGCATAAAGTATGCATAATGGTGTACCTAGAGTGACCTCTAAGAATGGTGATTATATTGTATTAGTATCACCTTAACTTAAGGACCAACATAAAGGGAGGAGACTCATGTTCCGCTTATTGTTGAACCTACTGCGGCATAGAGTCACCTACCGAT

TTCTTGTGGTACTTTGTGCTGCCCTTGGGTACGCATCTCTTACTGGAGACCTCAGTTCACTGGAGTCTGTCGTTTGCTCTATACTCACTTGTAGCGATTAGGGTCTTCCTGACCGACTGATGGCTCACCGAGGGATTCAGCGGTATGATTGCATCACACCACTTCATCCCTATAGAGTCAAGTCCTAAGGTATACCCATAAAGAGCCTCTAATGGTCTATCCTAAGGTCTATACCTAAAGATAGGCCATCCTATCAGTGTCACCTAAAGAGGGTCTTAGAGAGGGCCTATGGAGTTCCTATAGG

GTCCTTTAAAATATACCATAAAAATCTGAGTGACTATCTCACAGTGTACGGACCTAAAGTTCCCCCATAGGGGGTACCTAAAGCCCAGCCAATCACCTAAAGTCAACCTTCGGTTGACCTTGAGGGTTCCCTAAGGGTTGGGGATGACCCTTGGGTTTGTCTTTGGGTGTTACCTTGAGTGTCTCTCTGTGTCCCT

25

1 4 5 6 8 91

012 15 16 17

1

9

Functional depiction of T7 genome

26

Dunn & Studier (1983)Journal of Molecular Biology v166 p477

27

1 4 5 6 8 91

012 15 16 17

1

9

Functional depiction of T7 genome

28

14 56 891

012 1516 17

1

9

I have no idea what this genetic program will do

29

Wild-type T7 genes 2.8-3

-------------------2.8----------------->

...acgcaaagggaggcgacatggcaggttacggcgctaaaggaatccgaaa...

<--3-RBS---><----------------3-----------------

Natural DNA sequence

30

J.R. Koza et al.Automated Synthesis of Computational Circuits using GeneticProgramming,1997 IEEE International Conference on Evolutionary Computation

Evolved Circuit:

1. What does it do?2. How does it work?3. Why this design?

31

Design & build new genome?

32

Phage T7: Post-Synthesis

1. Design and construct any mutant or combinations thereof; characterize.

34

Post-Synthesis Genetics?

2. Reveal / confirm functions or demonstrate absence thereof.

1. Synthesize and assemble DNA fragments of length, L (say 1.5x max CDS length), offset so as to provide 3x coverage, only including known elements, deleting, scrambling, or otherwise destroying all unannotated sequence (i.e., simplons).

35

2. Recombine simplons back onto native genome, or into generic testbed (perhaps a generic deletion collection), screen.

0. Given a newly sequenced & annotated genome...

36

4. As a biproduct of preceding, produce full collection of native genetic elements: CDSs, regulatory elements, and so on.

37

40

“I think you're just ignoringthe facts.

I'm a geneticist, I write code, okay?

A, G, T, P in different combinations; hacking the human genome, okay?

I choose what I choose. Either your kidneys work or you grow six fingers.

I do that.”

Tom Sizemore, Red Planet (2000).

- Primary sequence assembly, annotation, analysis

- Functional primary networks (graphs)

- Secondary structure (2D physics)

- Tertiary structures, networks, dynamics (3D physics)

- Reverse engineering tools (for reproducing machines; time-scale decoupling of control systems; pattern forming systems)

- Post-synthesis biology tools (perturbation design)

Proposed Computing Priorities


ChallengeMake biology easy

to engineer.

OpportunityEnable all of biotechnology.



43

People

Tools

Platforms

Translation

45

People

Tools

Platforms

Translation

Over 3500 BioBrick DNA parts

Freely available*

Today

47

People

Tools

Platforms

Translation

Today, each project is Herculean

1) Malaria is a global problem,artemisinin offers a cure.

2) Jay Keasling’s team spent $25M to make artemisinin via biotechnology.

3) But artemisinin resistance is already occurring.

48

Must we always spend many years and $25M for each pressing

biotech project?

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Food Energy Environment Agriculture Health Chemicals Security

2004 DOE report lists 120 highvalue chemicals for biomanufacturing

Biotechnology is everywhereFo

od

Ener

gy

Env

ironm

ent

Agr

iculture

Hea

lth

Chem

ical

s

Secu

rity

$20B $14B$91B$3B$2.1B$2B$2.5B

NIH spends $1.5B just on manipulating DNA

AAAS 2008 Federal R&D Budget Analysis; R&D Magazine 2008; John Mulligan, Blue Heron

Total U.S. R&D spending by sector

50

Investment in open technology platforms non-existent

We need new tools

Cohen et al., PNAS, 1973

2006Production of the antimalarial drug

precursor artemisinic acid in engineered yeast

Ro et al., Nature, 2006

1973

Construction of biologically functional

bacterial plasmids in vitro

Lin et al., PNAS,1985

1985

Cloning and expression of the human

erthropoietin gene

51Genetic engineering basics unchanged past 30+ years

52

Synthetic biology = tools revolutionDNA Sequencing

Read Out the Genetic Code

Recombinant DNA

Basic “Cut” & “Paste”

Polymerase Chain Reaction

Amplify & Make Simple Changes

First

Gen.

Biotech=

...

Next

Gen.

Biotech

Adds

New

Tools

=

53

A public-benefit facility producing the parts, tools & standards powering biotechnology.

What is the BioFAB?

“Off-the-shelf genetic parts cut our project development times and overall costs by half.”

- Jack Newman, Founder & SVP Research, Amyris Biotechnologies

Canton, Labno & Endy

Nature Biotechnology v26:7, 787-93 July 2008



57

Mechanism & Function

A transcription factor (LuxR) that is active in the presence

of a cell-cell signaling molecule (3OC6HSL) is controlled by

a regulated operator (PLtetO-1). Device input is 3OC6HSL.

Device output is PoPS from a LuxR-regulated operator. If

used in a cell containing TetR then a second input such as

aTc can be used to produce a Boolean AND function.

BBa_F26203OC6HSL PoPS Receiver

Registry of Standard Biological Parts making life better, one part at a time

License: Public

Conditions (abridged)

Output: PoPS measured via BBa_E0240

Culture: Supplemented M9, 37ºC

Plasmid: pSB3K3

Chassis: MG1655

*Equipment: PE Victor3 multi-well fluorimeter

**Equipment: BD FACScan cytometer

1E01E1

1E21E3

1E4

GFP (arbitrary units)

Doublin

gs

20

38

56

74

92

High Input (1E -7 M 3OC6HSL)

92

1E01E1

1E21E3

1E4

Doublin

gs

20

38

56

74

Low Input(0 M 3OC6HSL)

GFP (arbitrary units)

Reliability**

Genetic: >92/>56 culture doublingsPerformance: >92/>56 culture doublings

(low/high input during propagation)

Transcriptional Output Demand (low/high input)

Nucleotides: 0 / 6xNt nucleotides cell-1 s-1

Polymerases: 0 / 1.5E-1xNt RNAP cell-1

(Nt = downstream transcript length)

Chassis: MC4100, MG1655, and DH5

Plasmids: pSB3K3 and pSB1A2

Devices: E0240, E0430 and E0434

BBa_F2620 Response Time: <1 minBBa_T9002 Response Time: 6±1 minInputs: 0 M (Low), 1E-07 M (High) 3OC6HSL

Pmax: 6.6 PoPS cell-1

K: 1.5E-09 M 3OC6HSLn: 1.6

Static Performance*

Pout =Pmax [3OC6HSL]n

Kn+ [3OC6HSL]n

0E+00 1E!10 1E!09 1E!08 1E!07 1E!06 1E!05 1E!04

0

100

200

300

400

500

600

[3OC6HSL] (M)

GF

P s

yn

the

sis

ra

te (

mo

lec

ule

s c

ell!1 s!1)

Population Mean

Colony Range

Hill Equation

0

1

2

3

4

5

6

7

8

Po

PS

ce

ll!1

Part Compatibility (qualitative)

Authors: Barry Canton Ania Labno

Updated: March 2008

Sig

nalin

g D

evic

es

http://p

art

s.m

it.e

du/r

egis

try/index.p

hp/P

art

:BB

a_F

2620

BBa_F2620

R0040 B0034 C0062 B0015 R0062

PLtetO-1 RBS luxR Term. Plux,R

Component Parts

!10 0 10 20 30 40 50

0

100

200

300

400

500

600

+ 3OC6HSL

Time (min)

GF

P s

yn

the

sis

ra

te (

mo

lec

ule

s c

ell!1 s!1)

GFP synthesis rate (Low Input)

GFP synthesis rate (High Input)

Polynomial Fit (High Input)

PoPS (High Input)

0

1

2

3

4

5

6

7

8

Po

PS

ce

ll!1

0E+00 1E!10 1E!09 1E!08 1E!07 1E!06 1E!05 1E!04

0

100

200

300

400

500

600

[AHL] (M)

GF

P s

yn

the

sis

ra

te (

mo

lec

ule

s c

ell!1 s!1)

C4HSL

C6HSL

3OC6HSL

C7HSL

C8HSL

3OC8HSL

C10

HSL

C12

HSL

0

1

2

3

4

5

6

7

8

Po

PS

ce

ll!1

Input Compatibility*

Dynamic Performance*



UnknownOur Lord's Candle (Yucca whipplei), Lakeview Mountains, 22 Apr 2006Smugmug.com

Unknownhttp://www.rootsweb.com/~usgenweb/mn/stearns/postcards/quarry.jpg

Washington National Cathedralhttp://www.cathedral.org/cathedral/discover/1930photos/5.shtml

http://www.cathedral.org/cathedral/discover/1930photos/5.shtml

http://www.cathedral.org/cathedral/discover/1930photos/5.shtml

http://www.nicmyers.com/media/hongkong_large_pano_nicmyers.jpg



http://en.wikipedia.org/wiki/Image:Eniac.jpg



http://en.wikipedia.org/wiki/Image:Apple_I.jpg



http://en.wikipedia.org/wiki/Internet



Apps

Tools

68

1. Info. theory & signal proc.2. Device design

7. Control & dyn. systems

5. Fab, CAD & EDA

6. Reverse engineering

3. Languages & grammars4. Standards & abstraction

69

70

Nagpal, Kondacs, Chang, Programming Methodology for Biologically-Inspired Self-Assembling Systems, in the AAAI Spring Symposium on Computational Synthesis: From Basic Building Blocks to High Level Functionality, March 2003.

Movie online via http://www.swiss.csail.mit.edu/projects/amorphous/Robust/cross.mpeg

71

1. Recombinant DNA

5. Abstraction

2. Polymerase chain reaction

3. DNA sequencing4. DNA construction

6. Standardization

- Advocacy for tools!!!

- Advocacy for open technology platforms.

- More tools, including: - Languages (e.g., for programming dynamic shapes) - CAD and EDA (what’s going into the synthesizers?) - Coding schemes (signatures, fail fast, et cetera)

- Theory supporting of design of understandable reproducing machines

- Tools that enable humanity (e.g., Games, Community)

Proposed Computing Priorities

Documents

Computing the Future of Biology & Biotechnologyarchive2.cra.org/ccc/files/docs/Endy_CCC_lite.pdf · ~5E9 tons plants (1% DNA, made up number) ~1E9 tons animals (1% DNA, made up number)