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:

• TRANS- repressor
• CIS- repressing

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

iGEM
2009
–
University
of
Bologna


O2


iGEM
2009
–
University
of
Bologna


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.



BASER

Best Sequence Research by Andrea and Elisa

iGEM
2009
–
University
of
Bologna


HOW BASER WORKS?

Star?ng
from
a
randomly
generated
sequence
(current
sequence);
 Conformity
test:
 a) more
than
5
adjacent
 repeats
of
the
same
 nucleo?de;

 b) restric?on
sites;

 c) RBS
sequences;


NO YES

iGEM
2009
–
University
of
Bologna
 BASER
replace
5
nucleto?des
randomly
(genera?on
of
new
sequence);
 Evaluate
score
of
new
sequence;


Add
RBS
at
3’
end


Evaluate
score
of
current
sequence;
 Score
of
new
 sequence
is
 beSer
than
 score
of
 current
 sequence?


New
sequence
 is
preserved
 Current
 sequence
is
 preserved


NO YES

iGEM
2009
–
University
of
Bologna


• BASER calculates a score for the current sequence:

1) the self score; 2) the genomic score; 3) the shifted score; How
BASER
calculates
the
score?


iGEM
2009
–
University
of
Bologna


Choose
of
a
CIS
sequence
 AACACAAACTATCACTTTAACAACACATTACATATACATTAAAATATTACAAAGAGGAGAAA
 (RBS
in
italic)


iGEM
2009
–
University
of
Bologna


CCTCTTTGTAATATTTTAATGTATATGTAATGTGTTGTTAAAGTGATAGTTTGTGTT
 with
a
7b‐long
RBS
cover
in
green
 Choose
of
the
TRANS
sequences
 CTTTGTAATATTTTAATGTATATGTAATGTGTTGTTAAAGTGATAGTTTGTGTT
 with
a
4b‐long
RBS
cover
in
green
underlined


iGEM
2009
–
University
of
Bologna


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


VIFluoR

iGEM
2009
–
University
of
Bologna


Par art Char t Characteriza acterization tion

• 
 Promoter
Strengths

• 
 Plasmid
copy
numbers

• 
 Influence
of
O2
operator

• 
 Interac?on
between
LacI
and
O2
operator


pSB3K3 pSB1A2

BBa_J23100 BBa_K07919 BBa_B0034 BBa_J0431 BBa_B0015 BBa_J23100 BBa_B0015 BBa_J23118 BBa_C0012 BBa_B0015

iGEM
2009
–
University
of
Bologna


Pr Promoter Str

• moter Strengths

engths BBa_J23118 BBa_J23118

(1429) (1429)

BBa_K079032 on pSB1A2

BBa_J23100 BBa_B0034

BBa_K079031 on pSB1A2

BBa_J23118

vs


BBa_J23100 BBa_J23100

(2547) (2547)

iGEM
2009
–
University
of
Bologna


Pr Promoter Str

• moter Strengths

engths

Methods
 ‐
DH5α
cells
 ‐
M9
medium
 ‐
37°
overnight


Imaging
Analysis
 ‐
VIFluoR


• Fluorimeter

Analysis


‐
Tecan
M200


BBa_J23118
 BBa_J23100


iGEM
2009
–
University
of
Bologna


Pr Promoter Str

• moter 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 pSB1A2 pSB1A2

(high cop (high copy) y)

vs


pSB3K3 pSB3K3

(lo (low/medium cop w/medium copy) y)

BBa_K201003 on pSB1A2

BBa_K201003 on pSB3K3

Methods
 ‐
DH5α
cells

 ‐
M9
medium
 ‐
37°
overnight


pSB3K3
 pSB1A2


Plasmid Cop Plasmid Copy Number y Numbers s

iGEM
2009
–
University
of
Bologna


Imaging
Analysis
 ‐
VIFluoR


• Fluorimeter

Analysis


‐
Tecan
M200


iGEM
2009
–
University
of
Bologna


Influence of Influence of O2 O2 BBa_K079032 BBa_K079032

(O2 a (O2 absent) bsent)

vs
BBa_K201001

BBa_K201001

(O2 pr (O2 present) esent)

BBa_K079032 on pSB1A2

BBa_K201001 on pSB1A2

Methods
 ‐
DH5α
cell

 ‐
M9
medium
 ‐
37°
overnight


iGEM
2009
–
University
of
Bologna


• Fluorimeter

Analysis


‐
Victor
2


pSB1A2


Influence of Influence of O2 O2

iGEM
2009
–
University
of
Bologna


Positiv

• sitive Contr

e Control of

• l of T

Testing Cir esting Circuit cuit

BBa_K201002 on pSB3K3

BBa_K201001 on pSB1A2

iGEM
2009
–
University
of
Bologna


Imaging
Analysis
 ‐ 
VIFluoR
 ‐ 
several
images
 ‐ 
>60
bacteria/image


IPT IPTG induction: Sta G induction: Static R tic Response esponse

Methods
 ‐
DH5α
cells
 ‐
M9
medium
 ‐ 
37°
overnight
 ‐ 
several
IPTG
levels


iGEM
2009
–
University
of
Bologna


Fluorimeter
Analysis
 ‐
Tecan
M200
 ‐ 
Dilu?on
to
OD=0.1
 ‐ 
1°
sample:
No
IPTG
 ‐ 
2°
sample:
IPTG
100μM


• Growth
Curve

• Fluorescence


• 


Methods
 ‐
DH5α
cell

 ‐
M9
medium
 ‐ 
37°
overnight
 ‐ 
No
IPTG


IPT IPTG induction: Dynamic R G induction: Dynamic Response esponse

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


From
Experimental
Measurement


iGEM
2009
–
University
of
Bologna


PARAMETERS ASSIGNMENT

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


We
fiSed
experimental
data
in
order
to
iden?fy
LacI‐O2
dissocia?on
constant
and
 LacI‐IPTG
dissocia?on
constant


STATIC IPTG INDUCTION

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.


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


CONCLUSIONS

Characterize
or
improve
an
exis?ng
BioBrick
Part
or
Device
and
enter
 this
informa?on
back
on
the
Registry.

 Help
another
iGEM
team:

 Cloned
and
sent
the
BioBrick
BBa_K201002
to
the
UNIPV‐Pavia
iGEM
 team.


iGEM
2009
–
University
of
Bologna


HUMAN PRACTICE- SHARING

We realized:

• An Online Survey
• An Information Booklet

iGEM
2009
–
University
of
Bologna


ON-LINE SURVEY RESULTS

• General lack of knowledge about Synthetic Biology
• Most people expressed curiosity about Synthetic Biology and iGEM
• After reading the booklet, great part of the respondent recognized

the importance of a responsible and conscious use of Synthetic Biology

iGEM
2009
–
University
of
Bologna


Total
respondents:
484


Acknowledgements

• University of Bologna
• Ser.In.Ar Cesena
• Cultural Association San Sebastiano

Instructors: Silvio Cavalcanti, Francesca Ceroni, Emanuele Domenico Giordano, Alejandro Hochkoeppler, Marco Caprini

iGEM
2009
–
University
of
Bologna


iGEM
2009
–
University
of
Bologna