In Vitro Construction of Glucose-sensitive Drug Synthesis...
Transcript of In Vitro Construction of Glucose-sensitive Drug Synthesis...
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In Vitro Construction of
Glucose-sensitive Drug Synthesis System
September 27th, 2015
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Team
Prof. Tiangang LiuWuhan UniversitySchool of Pharmaceutical Sciences
WHU-pharm
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1. Background
2. Design
3. Results
4. Outlook
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Background
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 WorldCancer Report, Lyon, IARC Nonserial Publication
A leading cause of death worldwide
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Background
Anti-tumor Drugs
Cytotoxicity
low specificity
N
O
O
H2N
H3C
OO
ONH2
H
H
CH3
NH
Mitomycin
HNP ON O
Cl
Cl
H2O
CyclophosphamideCyclophosphamide
HN
NH
F
O O
5-Fluorouracil
Immunosuppresion
Vomiting
Diarrhea
Hair loss
Infertility
……
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Background
Increased glucoseconsumption
Tumor Cells
Low O2 level
How to discriminate tumor cells from normal cells?
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立题依据Background
Glucose Detection
Drug Synthesis
In vitro assayset-up
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立题依据Design Glucose
pyruvate
lactate
NADH
NAD
P ADP+
ATPcharge
ATP ADP AMP cAMP
Plac
Glucose lactose
Low glucose concentration
High cAMP concentration
Transcroption on
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立题依据
Glucose sensing system
In vitro transcription and translation system
Require a living organism
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立题依据Glucose
pyruvate
lactate
1. Glk2. Pgi3. PfkA4. FbaA5. TpiA6. Pgk7. GpmA8. Eno9. PykF10. GapA
11.LdhNADH
NAD
ADP
ATP
P +
cyaA
cAMP
cpdA
AMP
ADK
H2O+cAMP AMP𝒄𝒑𝒅𝑨ATP+AMP 2ADP𝑨𝑫𝑲
P P+
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立题依据
Glucose sensing system
In vitro transcription and translation system
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E.coli RNA polymeraseCore enzyme
Mixture of 20 amino acids linking on theircorresponding tRNA
NTPs
Energy regeneration system
Mixture of different parts of rRNA
CRP sensitive Report function
𝛼𝛼𝛽𝛽′𝜔
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Wait for an hour
plasmid DNA
Use directly forprotein synthesis
T7 S30 ExtractContains components forCoupled transcription &translation
Add plasimd containingDNA sequence of proteinof interest under the T7promoter and RBS
E.coli RNApolymeraseCore enzyme
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Results Purification of Enzymes
1 2 3 4 5 6 7 8 9 10
Enzyme Glk Pgi PfkA FbaA TpiA Pgk GpmA
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
SDS-PAGE result of glycolysis enzymes
Enzymes:A. Enzymes for glycolysis (9 enzymes)B. Lactate dehydrogenase (LDH)
Glucose
Pyruvate Lactate
2ADP
2ATP
NAD+
NADHA
B
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In vitro transcription and translation mix
(cAMP)
E.Coli RNA polymerase core enzyme (𝛼𝛼𝛽𝛽′𝜔)CRP
pSB1C3-BBa_J04450
In vitro transcription and translation(positive control and negative control)
Results
System components
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With cAMP(P)Without cAMP(N)
In vitro transcription and translation(positive control and negative control)
Results
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In vitro transcription and translation
There is esterase in the cell extract system and hydrolysis cAMP
The CRP protein is not functional
The amount of fluorescence is too low to detectIncrease the amount of plasmid we add to see if there is a increased intensity
Using PURExpress system instead of cell extract to rule out the celluar esterase
Repurify CRP protein to rule out the CRP problem
Results
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ADP
ATPcyaA
cAMP
AMP
cpdA
ADK
Future work
d[ATP]/dt=[ADP]*[glucose]-2*[ATP]+[ADP]*[ADP]-[AMP]* [ATP]
d[ADP]/dt=-2*[ADP]*[ADP]+[ATP]-[ADP]+2*[AMP]*[ATP]
d[AMP]/dt=0.01*[cAMP]-[ATP]*[AMP]+[ADP]*[ADP]
d[cAMP]/dt=-0.01*[cAMP]+[ATP]
d[Glucose]/dt=-[ADP]*[Glucose]
v(cpdA)=10 v(cpdA)=5
v(cpdA)=1 v(cpdA)=0.5
cAMP AMP
GlucosecAMP
GlucosecAMP
GlucosecAMP
GlucosecAMP
v(cpdA)=0.1GlucosecAMP
cAMP
glucose
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Future work
ATPcyaA
cAMP
AMP
cpdA
ADK
ADP 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
Set up in vitro assay:ADP+ADK+cyaA+cpdA& cAMP assay
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AckowndgementInstructor: 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
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Thanks for your attention.