Wearestudentsofhighschoolandfirstsemesters ofcollege. - - PowerPoint PPT Presentation

• We
are
students
of
high
school
and
first
semesters

• f
college.

• Under
the
guidance
of
PhD.
Raúl
Cuero,
who
is
our


mentor.



• Dr.
Cuero
founded
the
IPOC


Name:
Ruiz
snowcapped
mountain
 Height:
5.389
m
(17,784
@.)
 Extension:
58.300
ha


• The
world
is
running
out
of
potable
water.

• Device
detects
ions
of
sea
water
and






separate
them
by
breaking
the
bonds
 



of
the
sea
water
molecules.


• More
available
potable
water.

• CONSTRUCTION
OF
NEW
PARTS!



RNA
switch
/
Cytochrome
C
/
Ion
Channels
/
Proteins


Manizales


AtlanZc
 Ocean


Pacific
 Ocean


Goals:


• Purify
sea
water

• Use
the
neural
and
E‐nose
algorithms
to


experimentally
analyze
data
and
to
 further
predict
addiZonal
unknown
 informaZon


DEVICE
 COMPONENTS


Chassis:



• Geobacillus
stearotermophilus


(Thermopile
Bacterium)


Promoters:



• E.
Coli

• T7
Promoter

• Iron
(Fe
Cuero)


Proteins:



‐Chloride



‐Sodium


Ion
Channels:



‐TonB





‐ExbB


Cytochrome
C:



RBS:
 Stop:


Reporter:


• Cyan
Reporter


RNA
Switch:
 ON


OFF


ON OFF

¿How
does
our
 device
works?


Control


NADH

dehydrogenase
 Cytochrome
C
 NADH
 Electrons
 Ubiquinone
 Na
molecule
 Cl
molecule


NADH

 dehydrogenase
 Ubiquinone
 Cytochrome
 C


Electrons


OXIDATION


e Hydrogenions
 ATP
 synthase



NADH
 dehydrogenase


NADH


Ubiquinone
 P P P P P P A A P P A

P P A P

Devices


• Basic
device

• Secondary
device


PBSK
II

 Cyan
Reporter


ConstrucZng
the
device


Amp


PBSK
II

 Chloride
 PBSK
II

 Sodium


Amp
 Amp
 RBS
 Reporter
 Stop
 Na
Channel
 Cl
Channel


PBSK
II

 Cyan
Reporter


ConstrucZng
the
device


Amp


PBSK
II

 Chloride
 PBSK
II

 Sodium


Amp
 Amp
 RBS
 Reporter
 Stop
 Na
Channel
 Cl
Channel


PBSK
II

 Cyan
Reporter


ConstrucZng
the
device


Amp
 RBS
 Reporter
 Stop
 Cl
Channel
 Na
Channel
 T7


RNA
SWITCH
ON


O N

ParZal
 Molecule
 Expression


O N

OFF RNA
Switch
 ON
 RNA
Switch
 ON
 RNA
Switch
 OFF
 Complete
 Molecule
 Expression


Results


DNA
ConcentraFons:


Devices
 DNA
ConcentraFon
(ng/ µl)


Device
1
(Final
LigaZon)
 1107
 Control
Device
 917


Devices
 OpFcal
Density


Device
1
(Final
LigaZon)
 0,4
 Control
Device
 0,4


Gel
from
PCR
 LigaZon
of
NaCl
with
 desinged
primers


Bioassays
 ConcentraZon
of
NaCl
and
MgCl


• n
the
Culture
medium


0%
 100%
 200%


100%
=

 24
g
of
NaCl

 +

 5
g
of
MgCl2


Parameters
to
measure
device
 efficiency


• Salt
concentraZon
in
the
medium

• DNA
concentraZon

• Bacterial
growth

• ATP
producZon

• Fluorescence
intensity

• RNA
concentraZon


Main
units
of
our
project


Sea‐water
 Device


PBSK
II


Neural
Network


SALT
[
]
 OpFcal
density


NaCl


E‐nose


ComputaZonal
modeling



• Is
used
in
the
project
to
complement
the


laboratory
work.
With
this
tool
we
will
predict
 which
device
is
opZmal.


• Two
systems
are
used
to
increase
their


efficiency
and
to
reduce
its
error:


• E‐nose


• Neural
model


The
basic
equaFons
for
the
E‐nose
 algorithm:


Size
of
the
sequence
 CombinaFon
 LocaFon
 Distance

 Number
of
Parts
 LocaFon
regarding
RBS
 Type
of
chassis
 Expression
of
proteins
 RestricFon
sites
 Type
of
primer
 Type
of
enzyme
 Expression
of
reporter
 Salts
ConcentraFon
 Presence
of
F
 Presence
of
O2
 Presence
of
Ca

 Presence
of
Zn
 Presence
of
Mg
 Volume
 ATP
 [DNA]

ConcentraFon
 Device
ConcentraFon
 Time
of
exposiFon
 Fluorescence
 E X T R I N S I C 
 I N T R I N S I C 
 Type
of
vector
 OpFcal
 density
 Salt
 concentraFon
 BLACK
 BOX


Size
of
the
sequence
 CombinaFon
 LocaFon
 Distance

 Number
of
Parts
 LocaFon
regarding
RBS
 Type
of
chassis
 Expression
of
proteins
 RestricFon
sites
 Type
of
primer
 Type
of
enzyme
 Expression
of
reporter


OPTICAL
 DENSITY
 [
SALT
]


CONCENTRATION


Salts
ConcentraFon
 Presence
of
F
 Presence
of
O2
 Presence
of
Ca

 Presence
of
Zn
 Presence
of
Mg
 Volume
 ATP
 [DNA]

ConcentraFon
 Device
ConcentraFon
 Time
of
exposiFon
 Fluorescence
 I N T R I N S I C 
 E X T R I N S I C 


HypotheZcal
Model
PredicZon
To
Determine
The
 Importance
Of
Variables



Different
possible
model
results
 based
on
theoricall
data
with
a
 0.4
standard
deviaZon.


Cl
 Na
 Ion
channels
 Cytochro me
 RNA
swicth
 SeparaFon

 PredicFon


0
 0
 0
 0
 0
 0
 0,00
 1
 0
 0
 0
 0
 0,05
 0,00
 0
 1
 1
 1
 1
 0,1
 0,11
 1
 0
 1
 1
 1
 0,1
 0,13
 1
 0
 1
 0
 1
 0,1
 0,07
 1
 1
 0
 1
 0
 0,15
 0,22
 1
 1
 0
 0
 1
 0,2
 0,25
 1
 1
 0
 0
 0
 0,45
 0,42
 1
 1
 0
 1
 1
 0,5
 0,48
 1
 1
 1
 0
 0
 0,75
 0,77
 1
 1
 1
 0
 1
 0,75
 0,75
 1
 1
 1
 1
 0
 0,95
 0,94
 1
 1
 1
 1
 1
 1
 1,00


0
 0.2
 0.4
 0.6
 0.8
 1
 1.2
 1
 2
 3
 4
 5
 6
 7
 8
 9
 10
 11
 12
 13
 Series1
 Series2


HypotheZcal
Model
PredicZon
To
Determine
The
 Importance
Of
Variables



0.36
 0.37
 0.38
 0.39
 0.4
 0.41
 0.42
 RNA
 Ion
Channels
 Cytochromes


Part
Removed
 Calculated
error
 RNA
 0,415502626
 Ion
Channels
 0,406685639
 Cytochromes
 0,378694109


Conclusions


• All
the
parts
were
assembled
and
the
device


was
fully
assembled.


• Ion
Channels
and
Cytochrome
C
increase
the


performance
 of
 the
 device
 (enhance
 the



































 redox
potenZal).


• ComputaZonal


and
 biological
 work
 complement
each
other.


• We
are
currently
running
bioassays.