WHATS T-REX? Control the synthesis of any protein of interest - PowerPoint PPT Presentation

WHAT’S T-REX? • Control the synthesis of any protein of interest • Silence the protein expression faster than using classic regulated promoters iGEM
2009
–
University
of
Bologna 


This device is composed of two BioBricks: • CIS- repressing • TRANS- repressor iGEM
2009
–
University
of
Bologna 


• Transcription of the target gene yields a mRNA strand; • The mRNA with the CIS sequence at 5' end, is available for translation. iGEM
2009
–
University
of
Bologna 


• When the promoter controlling the TRANS coding sequence is active its transcript binds with the CIS mRNA. • This RNA duplex prevents ribosomes from binding to RBS, thus silencing protein synthesis. iGEM
2009
–
University
of
Bologna 


TESTING CIRCUIT O2
 iGEM
2009
–
University
of
Bologna 


BASER Best Sequence Research by Andrea and Elisa 1) Maximal
free
energy
in
the
secondary
structure,
reducing
the
 probability
of
its
intra‐molecular
annealing;
 2) Minimal
unwanted
interac?ons
with
genomic
mRNA;

 3) Minimal
probability
of
par?al/shiEed
hybridiza?on
with
 complementary
strands.

 iGEM
2009
–
University
of
Bologna 


HOW BASER WORKS? Star?ng
from
a
randomly
generated
sequence
(current
sequence);
 Conformity
test:
 a) more
than
5
adjacent
 NO repeats
of
the
same
 nucleo?de;

 b) restric?on
sites;

 c) RBS
sequences;
 YES iGEM
2009
–
University
of
Bologna 


Add
RBS
at
3’
end
 Evaluate
score
of
current
sequence;
 BASER
replace
5
nucleto?des
randomly
(genera?on
of
new
sequence);
 Evaluate
score
of
new
sequence;
 Score
of
new
 Current
 YES sequence
is
 NO New
 sequence
 beSer
than
 sequence
is
 is
preserved
 score
of
 preserved
 current
 sequence?
 iGEM
2009
–
University
of
Bologna 


How
BASER
calculates
the
score?
 • BASER calculates a score for the current sequence: 1) the self score; 2) the genomic score; 3) the shifted score; iGEM
2009
–
University
of
Bologna 


Choose
of
a
CIS
sequence
 AACACAAACTATCACTTTAACAACACATTACATATACATTAAAATATTAC AAAGAGGAGAAA 
 (RBS
in
 italic )
 iGEM
2009
–
University
of
Bologna 


Choose
of
the
TRANS
sequences
 CCTCTTT GTAATATTTTAATGTATATGTAATGTGTTGTTAAAGTGATAGTTTGTGTT 
 with
a
7b‐long
RBS
cover
in
green
 CTTT GTAATATTTTAATGTATATGTAATGTGTTGTTAAAGTGATAGTTTGTGTT 
 with
a
4b‐long
RBS
cover
in
green
underlined
 iGEM
2009
–
University
of
Bologna 


VIFluoR Morphology:
 
‐
Eccentricity
[0,1];
 
‐
Area
[min,max];
 Focus:
 
‐
Clustering;
 








‐
High
fluorescence;
 








‐
High
cell
number;

 Output:
for
each
bacterium
the
area
in
pixels
and
the
fluorescence
 iGEM
2009
–
University
of
Bologna 


Par art Char t Characteriza acterization tion BBa_J23118 BBa_C0012 BBa_B0015 BBa_J23100 BBa_B0034 BBa_B0015 BBa_J23100 BBa_B0015 pSB3K3 BBa_K07919 BBa_J0431 pSB1A2 • 
 Promoter
Strengths
 • 
 Plasmid
copy
numbers
 • 
 Influence
of
O2
operator
 • 
 Interac?on
between
LacI
and
O2
operator
 iGEM
2009
–
University
of
Bologna 


Promoter Str Pr omoter Strengths engths BBa_J23100 BBa_J23100 BBa_J23118 BBa_J23118 vs 
 (2547) (2547) (1429) (1429) BBa_B0034 BBa_B0015 BBa_J23100 BBa_B0034 BBa_B0015 BBa_J23118 BBa_J04031 BBa_J04031 BBa_K079031 on pSB1A2 BBa_K079032 on pSB1A2 iGEM
2009
–
University
of
Bologna 


Promoter Str Pr omoter Strengths engths 
 Methods
 • ‐
DH5α
cells
 ‐
M9
medium
 ‐
37°
overnight
 BBa_J23118
 
 Imaging
Analysis
 • ‐
VIFluoR 
 • 

 
Fluorimeter

Analysis
 BBa_J23100
 ‐
Tecan
M200 
 iGEM
2009
–
University
of
Bologna 


Pr Promoter Str omoter Strengths engths 
 OD/Fluorescence
over?me
analysis
from
OD=0.1au
 • ‐ 
Growth
Curve
 ‐ 
Fluorescence

 ‐ 
Fluorescence/OD
ra?o
 iGEM
2009
–
University
of
Bologna 


Plasmid Cop Plasmid Copy Number y Numbers s pSB3K3 pSB3K3 pSB1A2 pSB1A2 vs 
 (lo (low/medium cop w/medium copy) y) (high cop (high copy) y) BBa_J23118 BBa_B0034 BBa_B0015 BBa_J23118 BBa_B0034 BBa_B0015 BBa_E0040 BBa_E0040 BBa_K201003 on pSB1A2 BBa_K201003 on pSB3K3 iGEM
2009
–
University
of
Bologna 


Plasmid Copy Number Plasmid Cop y Numbers s Methods
 
 • ‐
DH5α
cells

 ‐
M9
medium
 ‐
37°
overnight
 pSB1A2
 
 Imaging
Analysis
 • ‐
VIFluoR 
 • 

 
Fluorimeter

Analysis
 ‐
Tecan
M200 
 pSB3K3
 iGEM
2009
–
University
of
Bologna 


Influence of O2 Influence of O2 vs 
 BBa_K201001 BBa_K201001 BBa_K079032 BBa_K079032 (O2 present) (O2 pr esent) (O2 absent) (O2 a bsent) BBa_J23100 BBa_B0034 BBa_B0034 BBa_B0015 BBa_B0015 BBa_J23100 BBa_K07919 BBa_E0040 BBa_E0040 BBa_K079032 on pSB1A2 BBa_K201001 on pSB1A2 iGEM
2009
–
University
of
Bologna 


Influence of Influence of O2 O2 
 Methods
 • ‐
DH5α
cell

 ‐
M9
medium
 ‐
37°
overnight
 pSB1A2
 • 

 
Fluorimeter

Analysis
 ‐
Victor
2 
 iGEM
2009
–
University
of
Bologna 


Positiv ositive Contr e Control of ol of T Testing Cir esting Circuit cuit BBa_J23100 BBa_J23118 BBa_C0012 BBa_B0015 BBa_B0034 BBa_B0015 BBa_B0034 BBa_k07919 BBa_K201002 on pSB3K3 BBa_K201001 on pSB1A2 iGEM
2009
–
University
of
Bologna 


IPTG induction: Sta IPT G induction: Static R tic Response esponse Methods
 
 • ‐
DH5α
cells
 ‐
M9
medium
 ‐ 
37°
overnight
 ‐ 
several
IPTG
levels
 Imaging
Analysis
 
 • ‐ 
VIFluoR
 ‐ 
several
images
 ‐ 
>60
bacteria/image
 iGEM
2009
–
University
of
Bologna 


IPT IPTG induction: Dynamic R G induction: Dynamic Response esponse 
 Fluorimeter
Analysis
 • Methods
 
 • ‐
Tecan
M200
 ‐
DH5α
cell

 ‐ 
Dilu?on
to
OD=0.1
 ‐
M9
medium
 ‐ 
1°
sample:
No
IPTG
 ‐ 
37°
overnight
 ‐ 
2°
sample:
IPTG
100μM
 ‐ 
No
IPTG
 • 


Growth
Curve
 • 


Fluorescence

 iGEM
2009
–
University
of
Bologna 


MATHEMATICAL MODEL • 
Transcrip?on
and
transla?on
processes
were
considered
similar
to
a
second
order
 kine?cs,
like
an
enzyma?c
reac?on:
 iGEM
2009
–
University
of
Bologna 


MATHEMATICAL MODEL iGEM
2009
–
University
of
Bologna 


iGEM
2009
–
University
of
Bologna 


iGEM
2009
–
University
of
Bologna 


PARAMETERS ASSIGNMENT 
From
Literature
 iGEM
2009
–
University
of
Bologna 


PARAMETERS ASSIGNMENT From
Experimental
Measurement
 PROMOTER
RATIO
=1.2

 PLASMID
COPY
NUMBER
RATIO=4.6
 We
simulated
tes?ng
circuit
when
T‐REX
device
is
idle
(Ini?al
Trans‐DNA
=
0)
 iGEM
2009
–
University
of
Bologna 


LacI SIGMOIDAL REPRESSION CURVE iGEM
2009
–
University
of
Bologna 


STATIC IPTG INDUCTION We
fiSed
experimental
data
in
order
to
iden?fy
LacI‐O2
dissocia?on
constant
and
 LacI‐IPTG
dissocia?on
constant
 iGEM
2009
–
University
of
Bologna 


DYNAMIC IPTG INDUCTION Fiqng
of
the
100
µM
IPTG
dynamic
induc?on
with
?me‐varying
RNA
polymerase
 iGEM
2009
–
University
of
Bologna 


T-REX SIMULATION iGEM
2009
–
University
of
Bologna 


T-REX ST -REX STOR ORY We
didn’t
manage
to
get
the
final
circuit
because
we
didn’t
achieve

 the
assemblying
of
the
CIS
and
TRANS
parts
 Which
were
the
problems?
 1. Parts
are
only
100
bp
in
length:
 Quan?ty
problem,
due
to
purifica?on? 
 






*
P1010
death
gene
liga?on
protocol.
 2. Enzyme
efficency
is
lower
with
short
flanking
sequences:
 Were
our
diges?ons
effec?ve?


 






*
We
order
longer
PCR
primers
and
doubled
the
diges?on
?me.
 iGEM
2009
–
University
of
Bologna 


CONCLUSIONS Enter
informa?on
detailing
at
least
one
new
standard
BioBrick
Part
or
Device
in
 the
Registry
of
Standard
Parts
and
demonstrate
that
works
as
expected;

 Submit
DNA
for
at
least
one
new
BioBrick
Part
or
Device
to
the
Registry
of
 Parts.

 iGEM
2009
–
University
of
Bologna