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Building with light2015.igem.org/files/poster/TU_Darmstadt.pdf · 2015. 10. 8. · yjhG yagG yqhD...
1
TU Darmstadt 2015 Building with light Metabolic Engineering CadA Itaconic acid Glycolaldhyd xylC xylB Ethylene glycol Xylonic acid 2-dehydro- 3-deoxy- D-pentonate yjhG yagG yqhD TCA cis-Aconitat xylA Xylose PPP Glycolysis Glucose Xylitol GRE3 Xylose xylE in vivo XynA Silica-Tag GBD SH3 PDZ RuXyn1 aes in vitro Chemistry Policy & Practices Mechanical Part Xylitol Itaconic acid Polyethyleneglycol + Ethylene glycol Ethylene glycol Pre-polymer Monomer bricks Biosafety R N A - T e c h . C e l l - B i o l o g y cis-repressing RNA (crRNA) blocks hokD -gene no trans-activating RNA (taRNA) Production of HokD Release into the environment M o d e l i n g Xylose O OH OH OH OH M o l e c u l a r - B i o l o g y G e n e t i c s P r o t e i n - T e c h . B i o - C h e m i s t r y P o l y m e r - C h e m i s t r y S c e n a r i o P a n e l D i s c u s s i o n C o n s u l ti n g E x p e r t s M a t e r i a l s S c i e n c e O p t i c s E n g i n e e r i n g Xylan O O O O O O O O O OH - O2C H3CO HO HO OH HO O H3C O n O O OH OH O O HO H3CO Prom ta cr hokD RBS Constitutive transcription off Prom ta taRNA cr RBS hokD cr hokD RBS taRNA on hv P r o b l e m A p p l i c a ti o n S c e n a r i o T h e i G E M P r o c e s s S o l u t i o n O t h e r T e a m s S i d e P r o j e c t s E x p e r t s B i n a r y i npu t l a y e r Log i s t i c ou t pu t l a y e r e n c ode d b a s e p a i r s c o r i ng s s c o r e - ma t r i x Results of the FACS-measurements GFP under the araC - promotor araC-pBAD-GFP + glucose araC-pBAD-GFP + arabinose Fluorescence (arbitary units) Cell counts taRNA cr RBS hokD HokD taRNA/ hokD mRNA taRNA binds the crRNA 0.10 0.05 0.00 -0.05 0 50 100 150 200 - CadA + CadA Control cis-Aconitat A260 Reaction cycles Decarboxylic activity of cadA via pH indicator assay Polymer 75503b 16-36 RT: 2.19-4.93 : 21 : 4.82E5 T: c EI m 19.50-800.50 20 30 40 50 60 70 80 90 100 110 120 130 140 150 0 10 20 30 40 50 60 70 80 90 100 90.0 60.0 41.0 42.0 72.0 30.0 36.0 44.0 31.0 112.0 73.0 91.1 69.0 27.0 59.0 45.0 86.1 55.0 61.0 75503a #9-28 RT: 1.36-3.96 AV: 20 NL: 1.56E6 T: + c EI Full ms [19.50-800.50] 20 30 40 50 60 70 80 90 100 110 120 130 140 150 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 39.0 41.0 86.0 112.0 45.0 68.0 58.0 85.0 84.0 29.0 57.0 38.0 69.0 113.0 28.0 55.0 37.0 67.0 60.0 53.0 73.0 87.0 26.0 130.0 34.0 GC MS Itaconic acid molecule peak at 130u not detectable instable molecule, rapid decay biggest relative abundance at 41u washing with Tris-HCl Itaconic acid M + • Control g A A U U C G C G G C C G C U U C U A G A G a a c u a g a a u c a c c u c u u g c u u u u g g g u a a g a a a g a g g a g a U A C U A G a u g c g u a a a g g a g a a g a a c u u u u c a c u g g a g u u g u c c c a a u u c u u g u u g a a u u a g a u g g u g a u g u u a a u g g g c a c a a a u u u u c u g u c a g u g g a g a g g g u g a a g g u g a u g c a a c a u a c g g a a a a c u u a c c c u u a a a u u u a u u u g c a c u a c u g g a a a a c u a c c u g u u c c a u g g c c a a c a c u u g u c a c u a c u u u c g g u u a u g g u g u u c a a u g c u u u g c g a g a u a c c c a g a u c a u a u g a a a c a g c a u g a c u u u u u c a a g a g u g c c a u g c c c g a a g g u u a u g u a c a g g a a a g a a c u a u a u u u u u c a a a g a u g a c g g g a a c u a c a a g a c a c g u g c u g a a g u c a a g u u u g a a g g u g a u a c c c u u g u u a a u a g a a u c g a g u u a a a a g g u a u u g a u u u u a a a g a a g a u g g a a a c a u u c u u g g a c a c a a a u u g g a a u a c a a c u a u a a c u c a c a c a a u g u a u a c a u c a u g g c a g a c a a a c a a a a g a a u g g a a u c a a a g u u a a c u u c a a a a u u a g a c a c a a c a u u g a a g a u g g a a g c g u u c a a c u a g c a g a c c a u u a u c a a c a a a a u a c u c c a a u u g g c g a u g g c c c u g u c c u u u u a c c a g a c a a c c a u u a c c u g u c c a c a c a a u c u g c c c u u u c g a a a g a u c c c a a c g a a a a g a g a g a c c a c a u g g u c c u u c u u g a g u u u g u a a c a g c u g c u g g g a u u a c a c a u g g c a u g g a u g a a c u a u a c a a a u a a u a a U A C U A G U A G C G G C C G C U G C A G Computational Design of RNA-Riboswitches 1D 2D 3D W (i, j ) = min W (i +1,j ) W (i, j - 1) V (i, j ) min i<k<j {W (i, k)+ W (k +1,j )} V (i, j ) = min ΔG Hairpin (i, j ) ΔG Stack (i, j )+ V (i +1,j - 1) min i+1<j <j {ΔG Bulge (i, j, i +1,j )+ V (i +1,j )} min i<i <j-1 {ΔG Bulge (i, j, i ,j - 1) + V (i ,j - 1)} min i+1<i <j <j-1 {ΔG Interior (i, j, i ,j )+ V (i ,j )} min i<k<j-1 {W (i +1,k)+ W (k +1,j - 1)} +ΔG Multi (i, j ) . φ hidden (s) = tanh(s) ∈ [-1, 1]. φ out (s)= 1 1 - exp(-s) ∈ [0, 1]. f (s i )=c 1 ∑ l∈L φ(l, S i cr ) |L| + c 2 ∑ l∈L (1 - φ(l, S i ta )) |L| - c 3 MFE(S i cr ) - c 4 MFE(S i ta ), φ(l, S)= 1 , if position l is paired in S 0 , otherwise . Riboswitch Design Neural Folder (RsD-NerF) Computational Biology Logistic output layer Binary input layer Tanh hidden layer encoded base pair scorings score-matrix RNA- structure Modeling on our Wiki Find out more on our Wiki No glucose - araC is switched on Projector Mirror Client RaspberryPi 80 70 60 50 40 30 20 10 0 360 380 400 420 440 Intensity/Wavelength [mW m -2 nm - 1 ] Wavelength [nm] HG-Lamp Projector-Lamp UV-Lamp wavelength spectrum of the projectors lamp 410 Find out more about Synenergene Collaboration Find out more about GitHub Base plate Computer-aided design (CAD) Model Relative Abundance m/z
Transcript of Building with light2015.igem.org/files/poster/TU_Darmstadt.pdf · 2015. 10. 8. · yjhG yagG yqhD...
TU Darmstadt 2015Building with light
Metabolic Engineering
CadA
Itaconic acid
GlycolaldhydxylC
xylB
Ethylene glycol
Xylonic acid 2-dehydro-3-deoxy-
D-pentonate
yjhGyagG
yqhD
TCA
cis-Aconitat
xylA
Xylose
PPP Glycolysis
Glucose Xylitol
GRE3
XylosexylE
in vivo
XynA
Silica-Tag
GBD
SH3
PDZ
RuXyn1
aes
in vitro
Chemistry
Policy & Practices Mechanical Part
Xylitol
Itaconic acidPolyethyleneglycol
+
Ethylene glycol Ethylene glycol
Pre-polymer
Monomer bricks
BiosafetyRNA - Tech.
Cell - Biology
cis-repressing RNA (crRNA) blocks
hokD-geneno trans-activating
RNA (taRNA)
Production of HokD
Release into the environment
Modeling
Xylose
OOH
OH
OH
OH
Mole
cular - Biology
Genetics
Protein - Tech.
Bio-Chemistry
Polymer - Chemistry
Scen
ario
Pane
l Disc
ussion
Cons
ulting
Experts Materials Science
Optics
Engineering
Xylan
OO O
O O
O
O
OO
OH
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H3COHO
HO
OH
HO
O
H3C O
n
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OHO
O
HO
H3CO
Prom ta
crhokD
RBS
Constitutive transcription
off
Prom ta
taRNA
cr RBShokD
crhokD
RBS
taRNA
on
hv
ProblemApplication Scenario
The iGEM Process
Solution
Other Teams
Side Projects
Experts
Binary input layer
Logistic output layer
encoded base pair scorings
score-matrix
Results of the FACS-measurements GFP under the araC - promotor
araC-pBAD-GFP + glucose
araC-pBAD-GFP + arabinose
Fluorescence (arbitary units)
Cel
l cou
nts
taRNA
cr RBShokD
HokD
taRNA/ hokD mRNA
taRNA binds the crRNA
0.10
0.05
0.00
-0.050 50 100 150 200
- CadA
+ CadA
Controlcis-Aconitat
A260
Reaction cycles
Decarboxylic activity of cadA via pH indicator assay
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\\Thermo-8d83cadd\mat95 data\...\75503b 9/16/2015 17:17:36 Probe I 1 iGEM-TeamRT: 0.00 - 8.22
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.54.0
4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0Time (min)
10
20
30
40
50
60
70
80
90
100
Re
ati
e b
nda
nce
6.30
7.675.75 6.99
5.213.700.96 2.601.37
:8.88E7TI M 75503b
75503b 16-36 RT: 2.19-4.93 : 21 : 4.82E5T: c EI m 19.50-800.50
20 30 40 50 60 70 80 90 100 110 120 130 140 150m/
0
10
20
30
40
50
60
70
80
90
100
Re
ati
e b
nda
nce
90.0
60.0
41.0
42.072.030.0
36.044.031.0
112.073.0 91.169.027.0 59.045.0 86.155.0 61.0
75503a #9-28 RT: 1.36-3.96 AV: 20 NL: 1.56E6T: + c EI Full ms [19.50-800.50]
20 30 40 50 60 70 80 90 100 110 120 130 140 150m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
39.0
41.0
86.0
112.045.0
68.0
58.0 85.0
84.029.0
57.038.0 69.0113.028.0 55.037.0 67.060.053.0 73.0 87.026.0 130.034.0
GC MSItaconic acid molecule peak at 130u not detectable
instable molecule, rapid decay biggest relative abundance at 41u
washing with Tris-HCl
Itaconic acid
M+•
Control
gA
AU
U C GCGGCCGCUU C
UAGA
Gaac
ua
gaa
uc
acc
uc
uu
gcu
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ga a
ag
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ga g
aUA C
UA
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gaa
c u u u u ca c u
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ca
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uga
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auguuaaugggcacaaa
uuuucugucaguggagagggugaagguga
ugcaa
ca
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au
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caa
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cgu
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GC
GG
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UGC
AG
Computational Design of RNA-Riboswitches
1D
2D
3D
W (i, j) = min
W (i+ 1, j)
W (i, j − 1)
V (i, j)
mini<k<j
{W (i, k) +W (k + 1, j)}
V (i, j) = min
∆GHairpin(i, j)
∆GStack(i, j) + V (i+ 1, j − 1)
mini+1<j′<j
{∆GBulge(i, j, i+ 1, j′) + V (i+ 1, j′)}
mini<i′<j−1
{∆GBulge(i, j, i′, j − 1) + V (i′, j − 1)}
mini+1<i′<j′<j−1
{∆GInterior(i, j, i′, j′) + V (i′, j′)}
mini<k<j−1
{W (i+ 1, k) +W (k + 1, j − 1)}+∆GMulti(i, j)
.
φhidden(s) = tanh(s) ∈ [−1, 1].
φout(s) =1
1− exp(−s)∈ [0, 1].
f(si) =c1
∑l∈L φ(l,Si
cr)
|L|+ c2
∑l∈L(1− φ(l,Si
ta))
|L|− c3MFE(Si
cr)− c4MFE(Sita),
φ(l,S) =
{1 , if position l is paired in S0 , otherwise
.
1
Riboswitch Design
Neural Folder (RsD-NerF)
Computational Biology
Logistic output layer
Binary input layer
Tanh hidden layer
encoded base pair scorings
score-matrix
RNA-
stru
cture
Modeling on our Wiki
Find out more on our Wiki
No glucose - araC is switched on
Projector
Mirror
Client
RaspberryPi
80706050403020100 360 380 400 420 440
Inte
nsity
/Wav
elen
gth
[mW
m-2 n
m- 1 ]
Wavelength [nm]
HG-Lamp
Projector-LampUV-Lamp
wavelength spectrum of the projectors lamp
410
Find out more about Synenergene Collaboration Find out more about GitHub
Base plate
Computer-aided design (CAD) Model
Rel
ativ
e Ab
unda
nce
m/z
