In Vitro Construction of
Glucose-sensitive Drug Synthesis System
September 27th, 2015
Team
Prof. Tiangang Liu
Wuhan University
School of Pharmaceutical Sciences
WHU-pharm
1. Background
2. Design
3. Results
4. Outlook
Background
A leading cause of death worldwide
lung (1.59 million deaths)
liver (745 000 deaths)
stomach (723 000 deaths)
colorectal (694 000 deaths)
breast (521 000 deaths)
oesophageal cancer (400 000 deaths)
Bernard S. & Christopher W. 2014 World Cancer Report, Lyon, IARC Nonserial Publication
Background
H 2O
Cl
HN
P
N
O
O
Immunosuppresion
Anti-­tumor Drugs
low specificity
Cytotoxicity
O
Vomiting
Cl
Cyclophosphamide
Cyclophosphamide
Diarrhea
……
N
H
O
5-­Fluorouracil
Hair loss
O
Infertility
F
HN
O
O
H 2N
O
N
H 3C
O
Mitomycin
NH 2
CH 3
H
NH
H
Background
How to discriminate tumor cells from normal cells?
Low O2 level
Tumor Cells
Increased glucose
consumption
立题依据
Background
Glucose Detection
Drug Synthesis
In vitro assay
set-up
立题依据
Design
Plac
P
Glucose
charge
Glucose
+ ADP
Low glucose concentration
NAD
lactose
High
cAMP concentration
ATP
NADH
Transcroption on
pyruvate
ATP ADP AMP cAMP
lactate
立题依据
Require a living organism
Glucose sensing system
In vitro transcription and translation system
立题依据
Glucose
𝑨𝑫𝑲
1. Glk
𝒄𝒑𝒅𝑨
2ADP
HATP+AMP
O+cAMP
AMP
2.
Pgi
2
P + ADP
3. PfkA
4. FbaA
5. TpiA ADK
6. Pgk
AMP
NAD
7. GpmA
8. Eno
ATP 9. PykF
cpdA 10. GapA
cyaA
NADH
cAMP
+
lactate
11.Ldh
P P
pyruvate
立题依据
Glucose sensing system
In vitro transcription and translation system
CRP sensitive
E.coli RNA polymerase
Core enzyme
Report function
NTPs
Mixture of 20 amino acids linking on their
corresponding tRNA
Mixture of different parts of rRNA
Energy regeneration system
𝛼𝛼𝛽𝛽′𝜔
plasmid DNA
E.coli RNA
polymerase
Core enzyme
Wait for an hour
T7 S30 Extract
Contains components for
Coupled transcription &
translation
Add plasimd containing
DNA sequence of protein
of interest under the T7
promoter and RBS
Use directly for
protein synthesis
Results
Purification of Enzymes
Glucose
2ADP
NAD+
A
2ATP
NADH
Pyruvate
Lactate
B
Enzymes:
A. Enzymes for glycolysis (9 enzymes)
B. Lactate dehydrogenase (LDH)
SDS-PAGE result of glycolysis enzymes
1
2
3
4
5
6
7
8
9
10
Enzyme
Glk
Pgi
PfkA
FbaA
TpiA
Pgk
Gpm
A
Eno
PykF
GapA
Concentration (mM)
0.28
0.26
0.32
0.44
0.66
0.49
0.27
1.85
0.13
0.15
Results
In vitro transcription and translation
(positive control and negative control)
System components
(cAMP)
CRP
E.Coli RNA polymerase core enzyme (𝛼𝛼𝛽𝛽′𝜔)
pSB1C3-BBa_J04450
In vitro transcription and translation mix
Results
In vitro transcription and translation
(positive control and negative control)
With cAMP(P)
Without cAMP(N)
Results
In vitro transcription and translation
There is esterase in the cell extract system and hydrolysis cAMP
Using PURExpress system instead of cell extract to rule out the celluar esterase
The CRP protein is not functional
Repurify CRP protein to rule out the CRP problem
The amount of fluorescence is too low to detect
Increase the amount of plasmid we add to see if there is a increased intensity
Future work
glucose
v(cpdA)=5
v(cpdA)=10
ADP
ADK
Glucose
cAMP
Glucose
cAMP
d[ATP]/dt=[ADP]*[glucose]-­2*[ATP]+[ADP]*[ADP]-­[AMP]*
[ATP]
v(cpdA)=0.1
AMP
d[ADP]/dt=-­2*[ADP]*[ADP]+[ATP]-­[ADP]+2*[AMP]*[ATP]
Glucose
cAMP
cAMPd[AMP]/dt=0.01*[cAMP]-­[ATP]*[AMP]+[ADP]*[ADP]
AMP
cpdA
ATP
cyaA
cAMP
d[cAMP]/dt=-­0.01*[cAMP]+[ATP]
v(cpdA)d[Glucose]/dt=-­[ADP]*[Glucose]
=1
v(cpdA)=0.5
Glucose
cAMP
Glucose
cAMP
cAMP
Future work
ADP
ADK
AMP
cpdA
ATP
We have bought cAMP assay kit from Biovison:
That can directly measure the cAMP concentration
Set up in vitro assay:
ADP+ADK+cyaA
& cAMP assay
cyaA
cAMP
Set up in vitro assay:
ADP+ADK+cyaA+cpdA
& cAMP assay
Ackowndgement
Instructor: Prof. Tiangang Liu
Team advisor: Dr. Yi Liu
Molecular cloning & part construction: Da Di
Mathematical modeling: Da Di
Protein purification: Da Di, Yunfei Dai
Wiki design: Yunfei Dai
Poster design: Yunfei Dai, Haoxiang Qi
Graphic design: Haoxiang Qi
Thanks for your attention.