DavidsonMissouriWestern FoundationalAdvancesinBiology - - PowerPoint PPT Presentation

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DavidsonMissouriWestern FoundationalAdvancesinBiology andtheKnapsackProblem Whatistheknapsackproblem? ? 2kg 12kg 15kg 1kg 4kg

slide-1
SLIDE 1

Davidson
–
Missouri
Western


Foundational
Advances
in
Biology
 and
the
Knapsack
Problem


slide-2
SLIDE 2

What
is
the
knapsack
problem?


12
kg
 2
kg
 4
kg
 1
kg


?

15
kg


slide-3
SLIDE 3

What
is
the
knapsack
problem?


12
kg
 2
kg
 1
kg


15
kg


slide-4
SLIDE 4

Why
is
it
so
hard
to
solve?


?

87
kg


33
kg
 47
kg
 49
kg
 30
kg
 26
kg
 35
kg
 31
kg
 33
kg
 25
kg


slide-5
SLIDE 5

Why
is
it
so
hard
to
solve?


87
kg


30
kg
 26
kg
 31
kg


slide-6
SLIDE 6

FP
 TetAn
 FP
 TetAn
 FP
 TetAn


EXCISION INVERSION EXCISION

Choosing
Items
with
 the
Cre/Lox
System


slide-7
SLIDE 7

Excision

FP
 TetAn
 FP
 TetAn
 FP
 TetAn


Cre
 Cre
 Cre
 Cre


slide-8
SLIDE 8

FP
 TetAn
 FP
 TetAn
 FP
 TetAn


Excision

Cre
 Cre
 Cre
 Cre


slide-9
SLIDE 9

FP
 TetAn
 FP
 TetAn


Excision

Cre
 Cre
 Cre
 Cre


slide-10
SLIDE 10

FP
 TetAn
 FP
 TetAn


Cre
 Cre
 Cre
 Cre


Excision

slide-11
SLIDE 11

Inversion

FP
 TetAn
 FP
 TetAn
 FP
 TetAn


Cre
 Cre
 Cre
 Cre


slide-12
SLIDE 12

FP
 TetAn
 FP
 TetAn
 FP
 TetAn


Cre
 Cre
 Cre
 Cre


Inversion

slide-13
SLIDE 13

FP
 TetAn
 FP
 TetAn
 FP
 TetAn


Cre
 Cre
 Cre
 Cre


Inversion

slide-14
SLIDE 14

FP
 TetAn
 FP
 TetAn


Cre
 Cre


FP
 TetAn


Cre
 Cre


Inversion

slide-15
SLIDE 15

TetA
Pumps


Encoding
Item
Weights
with
Tet
Pumps


slide-16
SLIDE 16

Too
Few
Pumps
 Too
Many
Pumps
 Just
Right


slide-17
SLIDE 17

Too
Few
Pumps
 Too
Many
Pumps
 Just
Right


slide-18
SLIDE 18

#
of
TetA
Pumps
 Tetracycline
Concentration
 DEATH
 DEATH
 Too
Many
Pumps
 Too
Few
Pumps


slide-19
SLIDE 19

Cre


RFP
 TetA


slide-20
SLIDE 20

Building
a
Biological
Computer


lox
sites
=
choice
 TetR
=
weight
 nFP
=
object


tetA
 RFP


slide-21
SLIDE 21

Building
a
Biological
Computer


Lox
sites
=
choice
 TetR
=
weight
 nFP
=
object


tetA
 RFP


slide-22
SLIDE 22

Building
a
Biological
Computer


Lox
sites
=
choice
 TetR
=
weight
 nFP
=
object


tetA
 RFP


slide-23
SLIDE 23

Tools


  • The
Oligator

  • Veripart

  • Construct
Simulator

  • The
Optimus

  • Knapsack
Educational
Game

slide-24
SLIDE 24
  • Assists
in
de
novo
part
construction

  • Designs
oligos
for
annealing


The
Oligator


slide-25
SLIDE 25
  • Assists
in
the


analysis
of
 sequence
data


  • Compares
raw


sequence
to
 Registry


  • Outputs
matches


to
Registry
parts


slide-26
SLIDE 26

Construct
Simulator


  • Simulates
Cre
action

  • n
Knapsack


constructs



  • Demonstrates


recombination
of
lox
 sites


  • Outputs
fluorescent


protein
expression


slide-27
SLIDE 27

Codon
Optimization


  • Tet(A)
production
=
object
weight


– optimized
Tet(A)
=
heavy
object
 – deoptimized
Tet(A)
=
light
object
 – wt
Tet(A)
=
intermediate
object


slide-28
SLIDE 28

The
Optimus


  • Uses
codon
bias
to


alter
DNA
sequence
 for
a
given
 polypeptide



  • Frequently
used


codons
 (optimization)
or
 rarely
used
codons
 (deoptimization)
are
 selected


  • Possible
mechanism


to
regulate
gene
 expression


slide-29
SLIDE 29

Expanding
the
Cre/lox
System

  • Built
11
novel
lox
sites
that
exhibit
mutually
exclusive


recombination
 F
 R


slide-30
SLIDE 30

Characterization
of
recombination



  • 21
possible
lox
combinations

  • Each
lox
variant
is
color
coded
above


slide-31
SLIDE 31

Expanding
the
Cre/lox
System



  • 11
new
lox
variants
added
to
registry

  • Characterizing
valuable
foundational
tool
for


the
SB
community


  • Tested
pBad‐RBS‐Cre:
wrong
part


  • Rebuilt,
sequence
verified
pBad‐RBS‐Cre,
and


added
to
registry


pBAD
 RBS


Cre


slide-32
SLIDE 32

Foundational
Issues


  • Designed
&
modeled
Knapsack
Problem


computer


  • Initial
testing
revealed
anomalies


  • Two
options:

  • 1. Ignore


  • 2. Investigate


  • We
chose
to
explore
foundational
issues

slide-33
SLIDE 33

Five
Hard
Truths
for
Synthetic
Biology


  • 1. Many
of
the
parts
are
undefined

  • 2. The
circuitry
is
unpredictable

  • 3. The
complexity
is
unwieldy

  • 4. Many
parts
are
incompatible

  • 5. Variability
crashes
the
system


Kwok,
Roberta.
2010.
Five
Hard
Truths
for
 Synthetic
Biology.
Nature.
463:288‐290


slide-34
SLIDE 34

Five
Hard
Truths
for
Synthetic
Biology


  • 1. Many
of
the
parts
are
undefined

  • 2. The
circuitry
is
unpredictable

  • 3. The
complexity
is
unwieldy

  • 4. Many
parts
are
incompatible

  • 5. Variability
crashes
the
system


Kwok,
Roberta.
2010.
Five
Hard
Truths
for
 Synthetic
Biology.
Nature.
463:288‐290


slide-35
SLIDE 35

Observation
#1
 Circuitry
is
Unpredictable


RFP
 TetA
 RBS
 RBS
 RFP
 RBS
 RBS
 TetA


RFP+Tet
 Tet+RFP


RBS
 RFP


RFP


slide-36
SLIDE 36

Observation
#1
 Circuitry
is
Unpredictable


RFP
2nd

 RFP
1st

 RFP
alone
 RFP
2nd

 RFP
1st

 RFP
alone


slide-37
SLIDE 37

Observation
#2

 Many
Parts
are
Incompatible


AMP
 AMP
+

 IPTG
 TET
 TET
+

 IPTG


slide-38
SLIDE 38

Observation
#3
 Many
of
the
parts
are
undefined


Water
 LB


0
 24
 48
 72
 96
 120
 144
 pLac‐RBS‐RFP
 pLac‐RBS‐RFP
 AU
 Various
Constructs
(allowed
to
sit
in
water
or
broth)


Fluorescence
Measured
Over
a
Period
of
Time
in
Water
and
Broth


8:30am
 10:00am
 12:00pm
 2:00pm


Water
 LB
Broth


pLac‐RBS‐RFP


slide-39
SLIDE 39

Color

 Variation


Red
 Pink


Observation
#4
 The
Circuitry
is
Unpredictable


slide-40
SLIDE 40

Observation
#4
 The
Circuitry
is
Unpredictable


Pink
 Red


  • DNA
Sequence


identical


slide-41
SLIDE 41

Observation
#4


The
Circuitry
is
Unpredictable


Pink
Clone
 Red
Clone
 Red
Clone
 Pink
Clone


slide-42
SLIDE 42

A
 B
 C
 Ring


Observation
#5
 The
Circuitry
is
Unpredictable


slide-43
SLIDE 43

A
 C
 B
 Ring


slide-44
SLIDE 44
  • Designed
biological
computer
for
Knapsack


problem


  • Developed
5
software
tools
for
SB

  • Built
library
of
11
new
lox
sites

  • Built
42
basic
and
composite
parts

  • Rebuilt
Cre

  • Documented
foundational
issues


Accomplishments


slide-45
SLIDE 45

Foundational
Advances


  • Evidence
of
transcriptional
terminator
in
TetA

  • Quantified
interference
with
fluorescence


– Tetracycline

 – Water
vs.
media


  • Possible
epigenetic
regulation


  • Bacterial
community
dynamics


slide-46
SLIDE 46

Acknowledgements


  • NSF
Grants
DMS
0733952
and
0733955

  • HHMI
Grant
52006292

  • Davidson
College
Faculty
Study
and
Research
Grant,
Davidson


Research
Initiative,
and
James
G.
Martin
Genomics
Program


  • Missouri
Western
State
University
Foundation,
Academic
and


Student
Affairs,
and
Summer
Research
Institute


  • Genome
Consortium
for
Active
Teaching
(GCAT)

  • David
Bikard
from
2007
Paris
iGEM
Team

slide-47
SLIDE 47

Davidson
–
Missouri
Western


Foundational
Advances
in
Biology
 and
the
Knapsack
Problem


Kelia
Alfred,
Stacey
Holle,
Bridget
Janssen,
Curtiss
Lane,
 Steph
Meador,
Bri
Pearson,
Jamela
Peterson,
Anvi
Raina,
 Nitya
Rao,
Eugene
Shiu,
Tom
Shuman,
Jeff
Stevens,
 Stephen
Streb,
Jeska
Testa,
Danielle
Wagner