In Vitro Construction of

Glucose-sensitive Drug Synthesis System

September 27th, 2015

Team

Prof. Tiangang LiuWuhan UniversitySchool of Pharmaceutical Sciences

WHU-pharm

1.  Background

2.  Design

3.  Results

4. Outlook

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

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

……

Background

Increased glucoseconsumption

Tumor Cells

Low O2 level

How to discriminate tumor cells from normal cells?

立题依据Background

Glucose Detection

Drug Synthesis

In vitro assayset-up

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

立题依据

Glucose  sensing  system

In  vitro  transcription  and  translation  system

Require a living organism

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+

立题依据

Glucose  sensing  system

In  vitro  transcription  and  translation  system

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

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

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

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

With cAMP(P)Without cAMP(N)

In vitro transcription and translation(positive control and negative control)

Results

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

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

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

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

Thanks for your attention.