hong kong cuhk

47
The Chinese University of Hong Kong – iGEM 2010 Bacterial based storage and encryp2on device 1

Upload: industrieit

Post on 02-Apr-2015

479 views

Category:

Documents


2 download

TRANSCRIPT

Page 1: Hong Kong CUHK

The  Chinese  University  of  Hong  Kong  –  iGEM  2010  

Bacterial  based  storage  and  encryp2on  device  

1

Page 2: Hong Kong CUHK

Bacterial  based  informaAon  storage  device  

•  Bancro4’s  group  (2001)  Mount  Sinai  School  of  Meducube  

•  Yachie’s  group  (2007)  Keio  University  

 

2

Page 3: Hong Kong CUHK

This  year,  The  CUHK…  

•  True,  massively  parallel  bacterial  storage  system  

3

Page 4: Hong Kong CUHK

In  AddiAon…  

•  Encryp2on  module  with  DNA  shuffling  system  – Rci  system  

•  The  data  proof-­‐read  – Chechksum  

•  Strategy  deal  with  synthesis/sequencing  difficul2es  – Homopolymer,  repe22ve  sequence  

4

Page 5: Hong Kong CUHK

5

Page 6: Hong Kong CUHK

Basic    infrastructure  of  the  system  

Module-­‐based  

Processing  

Coding  System  

EncrypAon

MP  Storage  System

DecrypAon

6

Page 7: Hong Kong CUHK

MP  Storage  System

Quaternary  Number  System  

Encoding  table  

DNA  sequence  

Compression  

Coding System Coding  System  Encryp2on Decryp2on

7

Page 8: Hong Kong CUHK
Page 9: Hong Kong CUHK

Original message input

Converted to Quaternary number

Converted to DNA sequence

9

Page 10: Hong Kong CUHK

   Coding  -­‐  Compression  

• DEFLATE  —  a  compression  algorithm  

10

1.  Can  reduce  the  homopolymer  and  repeAAve  regions

2.  Can  store  more  informa2on

EncrypAon

MP  Storage  System

DecrypAon

Coding  System  

Page 11: Hong Kong CUHK

The length and repetitive sequence is greatly reduced

Homopolymer  

11

Page 12: Hong Kong CUHK

RepeAAve  Regions  

12

Page 13: Hong Kong CUHK

EncrypAon  

•  Examples:                                                    

13

Homologous  recombina2on

RACHITT

Coding  System  EncrypAon

MP  Storage  System

DecrypAon

Page 14: Hong Kong CUHK

Simulation Analysis

The  United  States  DeclaraAon  of  Independence  

8074  Characters!!!  

14

Page 15: Hong Kong CUHK

FragmentaAon  of  message  •  Larger  than  the  maximum  vector  inser2on  size  •  Limita2on  of  current  DNA  synthesis  technology            →  Split  the  message  into  different  parts  

15

How  do  you  deal  with  the  problem  of  posi2oning?

Postal Code

Page 16: Hong Kong CUHK

Storage  –  Massively  parallel    

Header  –  Locate  parAcular  data  fragment  of  the  message    

Analogy  to  the  hard  disk  :  4  address  units  

16 Low  Precision  

High  Precision  

Page 17: Hong Kong CUHK

AGAT   AGAC   AGTA   AGTG  

AGAT   AGTG   AGAT   AGAC  

AGAT   AGAC   AGTA   AGAG  Header  of  2nd    fragment  

Header  of  3rd        fragment  

Header  of    1st        fragment  

Header  of  4th  fragment   17

AGAT   AGAC   AGAG   AGCT  

Page 18: Hong Kong CUHK

18

0301   0302   0310   0313  

0301   0302   0303   0321  

0301   0313   0301   0302  

0301   0302   0310   0303  Header  of  2nd    fragment  

Header  of  3rd        fragment  

Header  of    1st  fragment  

Header  of  4th      fragment  

1  

2  

3  

4  

18

Page 19: Hong Kong CUHK

Only    18  cells!!!  

Capacity  

19

If  inser2on  size  per  cell  is  1kb……  

The  United  States  DeclaraAon  of  Independence  

Page 20: Hong Kong CUHK

Capacity  

The  United  States  DeclaraAon  of  Independence  requires    

20

Page 21: Hong Kong CUHK

DecrypAon  

21

sequencing

Iden2fica2on  of  repeat,  message,  checksum

Checksum  system

Coding  System  EncrypAon

MP  Storage  System

DecrypAon

Page 22: Hong Kong CUHK

Checksum  Mechanism  

22

F1   F3  F2   Checksum  

F2   F3  F1  

F1  F3  F2   Checksum  1  

Checksum  2  

CRC64

CRC64

Page 23: Hong Kong CUHK

23

Page 24: Hong Kong CUHK

To  prove  our  concept…  Coding  

(extended  ASCII  code)

EncrypAon  (message  +  rci  system)

Storage  (in  E.coli  DH5  α)

DecrypAon  (sequencing)

24

Page 25: Hong Kong CUHK

25

Page 26: Hong Kong CUHK

Message  

•  we  must  learn  to  live  together  as  brothers  or  perish  together  as  fools    <<code  form  Dr.  Mar2n  Luther  King,  Jr.,  a  prominent  leader  in  the  African  American  civil  rights  movement  >>  

Our message (70 characters)

DNA Encoding(280bp)

tools

eg. “tools”

DNA encoding: TGTATCGGTCGGTCGATGAG (20bp)

26

Page 27: Hong Kong CUHK

Structure  of  message  

•  Repeat  A  sequence  in  natural  shufflon  system  has  the  highest  inversion  frequency  

•  19bp  

         

27

Repeat A

Repeat A

Page 28: Hong Kong CUHK

Parts  designed

 

Rci  site-­‐specific  recombinase  (1155bp)  Synthesized  DNA  (rci  gene  sequence  of  E.  coli  (strain:  K-­‐12))

Message  gene  template  (438bp)  Synthesized  DNA

Rci  system  (1484bp)  • lac  promoter  • ribosome  binding  site  • rci  gene  • double  terminator  

28

Page 29: Hong Kong CUHK

29

IntegraAon  of  message  to  rci  system

Page 30: Hong Kong CUHK

•  Repeat  sequence  +  Message  +  Repeat  sequence  •  There  should  be  two  scenarios:  1. Inversion  of  message  2. No  change  of  original  message  •  Two  sets  of  primers  are  used  

ExpectaAon

Repeat+Message+Repeat Header Footer 5’ 3’ A

A’

PCR Can  be  amplified  and  sequenced

B

B’

PCR Can  be  amplified  and  sequenced

30

Page 31: Hong Kong CUHK

Results

• Inverted  and  original  message  were  found    

• No  loss  of  DNA  

Checksum  and  high  throughput  sequencing!!!

31

Page 32: Hong Kong CUHK

High  throughput  sequencing

•  Massively  parallel  sequencing  process  •  Mul2ple  copies  of  sequencing  products  (reads)  that  can  cover  a  par2cular  message  stored  within  the  DNA  

•  Enable  us  to  perform  a  majority  vo2ng  on  bases  for  which  quali2es  are  not  the  best  

32

Page 33: Hong Kong CUHK

33

Page 34: Hong Kong CUHK

To  summarize…  

34

•  Infrastructure  of  our  system  

   

•  Experimental  proof

Coding  System  

EncrypAon

MP  Storage  System

DecrypAon

Page 35: Hong Kong CUHK

Bio-­‐hard  disk

35

1  gram(wet  weight)  of  E.coli 2  TB  hard  disk

= 450

Storage  Hard  disk   2000GB  

1  gram  E.coli   900,000GB  

Page 36: Hong Kong CUHK

Rapid  &  Specific  access

•  Parallel  storage  system  

36

Insert  Header  &  Footer  in  every  message  fragment

Design  specific  probe  corresponding  to  Header

Pick  up  parAcular  message  from  pool  of  data

Targeted  sequencing

Page 37: Hong Kong CUHK

Future  ApplicaAon

•  Can  store  text,  images,  music,  movies……  

 •  Inser2on  of  barcodes  into  synthe.c  organisms  as  a  part  of  current  safety  protocols  to  dis2nguish  between  synthe2c  &  natural  organisms  

•  Store  addi2onal  informa2on:  

37

ABC

Page 38: Hong Kong CUHK

Acknowledgement

38

Page 39: Hong Kong CUHK

Further  InformaAon

•  Gyohda,  A.  &  Komano,  T.   (2000).  Purifica2on  and  Characteriza2on  of  the   R64   Shufflon-­‐Specific   Recombinase.   J.   Bacteriol.,   182   (10),  2787-­‐92.    

•  Gyohda,   A.,   Zhu,   S.,   Furuya,   N.   &   Komano,   T.   (2005).   Asymmetry   of  Shufflon-­‐specific   Recombina2on   Sites   in   Plasmid   R65   Inhibits  Recombina2on  between  Direct  sfx  Sequences.  J.  Biol.  Chem.,  281  (30),  20772-­‐9.    

 

hqp://2010.igem.org/Team:Hong_Kong-­‐CUHK

39

Page 40: Hong Kong CUHK

40

Page 41: Hong Kong CUHK

Q&A  

41

Page 42: Hong Kong CUHK

Inversion  frequency

1.  types  of  19-­‐bp  repeat  sequences  (repeat-­‐a>  repeat-­‐d>  repeat-­‐b  or  repeat-­‐c)    2.  distance  between  repeat  sequences  (distance  increases,  frequency  increases)  3.  DNA  sequences  surrounding  the  repeat  sequences(symmetric  repeat  sequence  increase  frequency)  

Page 43: Hong Kong CUHK

Inversion  frequency

4.    presence  of  HU  protein    (binding  of  HU  protein  to  DNA  might  facilitate  assembly  and/or  stabiliza2on  of  the  Rci-­‐DNA  complex  at  the  recombina2on  sites,  increases  frequency)  

5.  extent  of  DNA  supercoiling    (Inhibi2on  of  DNA  supercoilingà  decrease  Rci  ac2vityà  decrease  inversion  frequency)  

Page 44: Hong Kong CUHK

To  avoid  muta2on

-­‐    Reduce  reproduc2ve  cycle -­‐    Provide  favorable  condi2on -­‐    Move  on  to  eukaryotes,  make  use  of  eukaryotes’  proofreading  system(more  sophis2cated  DNA  repair  system)

Page 45: Hong Kong CUHK
Page 46: Hong Kong CUHK
Page 47: Hong Kong CUHK

Pacific  Bioscience  

•  Real  2me  •  Read  Length  :  1000  -­‐  10000bp  •  Single  Molecule  Sequencing  •  30  minutes  sequencing  process