DREAM FACTORY SHENZHEN UNIVERSITY CHINA Boston, USA 11/02/2014 - - PowerPoint PPT Presentation

ALKALINE CELLULASE DREAM FACTORY

SHENZHEN UNIVERSITY CHINA Boston, USA 11/02/2014

Background 1 Project & Design 2 Results 3 Human Practice 4

CONTENTS： Dream Factory

Alkaline Cellulase

BACKGROUND

PART 1：

 Paper recycling；Deinking  Environment and Sustainable Development Zh Zhipeng QI QIU

Alkaline Cellulase

Dream Factory

Chinese economy is developing rapidly However, our environment is deteriorating……

Dream Factory

Alkaline Cellulase

The polluted river in Rural China can be seen everywhere!

Dream Factory

Alkaline Cellulase

Simple and crude equipment

Waste paper recycling industry Use toxic chemicals during the deinking

Pour the waste water Into the river di directly!

Dream Factory

Alkaline Cellulase

The rise of synthetic biology provides a new way to solve this type of

environmental question.

We had made An incredible E.coli

• --- “Alkal

kalin ine e Cellulas lase Dre ream m Facto tory ry”

To reduce the use of harmful chemicals.

Project & Design

PART 1：

 Four systems of the Dream factory Yiti Yiting LU LUO

Alkaline Cellulase

Dream Factory

D r e a m F a c t o r y

Alkaline Cellulase

Alkaline Cellulase

• for the release of ink from the fiber

surface by partial hydrolysis of carbohydrate molecules Mdfa Antiporter

• resistance to alkaline wastewater

CCDB

Kil

• transportation system, enhance the

secretion of alkaline cellulase

• Suicide system

Dream Factory

Alkaline Cellulase

Cellulase

Mechanism of hydrolyzing internal β-1,4-β-bonds in cellulose catalyzed by endoglucanase(Brisan et al, 1998)

Dream Factory

Alkaline Cellulase

Alkaline Cellulase

• ------deinking

Release of ink from the fiber surface by partial hydrolysis of carbohydrate molecules. cellulose ink Alkaline Cellulase

endo-(1,4)-β-D-glucanase (EC 3.2.1.4)

D r e a m F a c t o r y

Alkaline Cellulase

Mdfa Antiporter OH

• OH
• OH
• OH
• OH
• OH
• OH
• OH
• Normal

bacteria OH

• OH
• OH
• OH
• OH
• OH
• OH
• OH
• Alkaline tolerance bacteria

Alterations of cell membrane Remodeling of metabolic patterns

Active inward transporters of protons

D r e a m F a c t o r y

Alkaline Cellulase

Mdfa Antiporter

Maintenance of a stable cytoplasmic pH under conditions of alkaline stress. Couple the inward movement of protons to the outward of Na+.

Edgar R, Bibi E. MdfA, an Escherichia coli multidrug resistance protein with an extraordinarily broad spectrum of drug recognition. J Bacteriol. 1997;179:2274–2280.

D r e a m F a c t o r y

Alkaline Cellulase

Kil gene1

For secretion of alkaline cellulase

Increase the permeability of cell membrane

1.Tong Q, Zhang H T, Yang Y G, Mediated by Kil protein of Escherichia coli secretory expression system (China Academy of Sciences in Shanghai Institute of Biological Engineering Research Center, Shanghai 200233)

D r e a m F a c t o r y

Alkaline Cellulase CCDB(control of celldivision death protein B)-Suicide system

Cell killing by the ccdB protein involves poisoning of DNA-topoisomerase II complexes. Suicide system can be induced by the addition of the chemical IPTG. ccdB induced by IPTG Gyrase

Dream Factory

Alkaline Cellulase

Integration FOUR PARTS

Alkaline line Cellulas lulase e Dream m Factory

• ry

Results

PART 3：

 Four systems of the Dream factory  Deinking Test Hu Huan Wei

Alkaline Cellulase

Dream Factory

Dream Factory

Alkaline Cellulase

Alkaline Cellulase Mdfa Antiporter Kil Protein ccdB Suicide System

Our Project has four parts：

Dream Factory

Alkaline Cellulase

Alkaline Cellulase

Figure 1. Using CMC-Na DNS method for detection of Alkaline Cellulase Figure 2. transparent zones produced by E.coli on CMC-containing solid plate

Cellulase can degrade cellulose.

1.use two methods to detect cellulase activity.

Dream Factory

Alkaline Cellulase

Alkaline Cellulase

■ without CaCl2

2.The optimum reaction pH and pH stability 3.Research on temperature stability

• f Alkaline Cellulase
• ptimum temperature is 50 ℃,

Ca2+ can improve stability.

◇ containing 5mM CaCl2

Optimum reaction pH is 9.0. Alkaline conditions with strong stability.

pH=9.0

Dream Factory

Alkaline Cellulase

Mdfa Antiporter

Growth contrast in LB plate medium with different pH.

Mdfa ：promoter+RBS+Mdfa control：promoter+RBS

Dream Factory

Alkaline Cellulase

Mdfa Antiporter

Chart of OD value of Mdfa device and control group in different pH liquid culture Mdfa device：promoter+RBS+Mdfa control：promoter+RBS

Dream Factory

Alkaline Cellulase

kil protein1

without kil Determination of extracellular enzyme activity .

Kil can enhance the secretion of alkaline cellulase .

with kil

1.Tong Q, Zhang H T, Yang Y G, Mediated by Kil protein of Escherichia coli secretory expression system (China Academy of Sciences in Shanghai Institute of Biological Engineering Research Center, Shanghai 200233)

0.018 0.042 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 LB PLATE WITHOUT KIL WITH KIL OD540 VALUE

Culture two days later OD540 value by DNS method measuring Alkaline Cellulase to test kil function

Dream Factory

Alkaline Cellulase

CCDB suicide system

Ou Our E. . co coli can‘t grow in the LB plate medium with IPTG

Dream Factory

Alkaline Cellulase

• Cellulase can successfully

degrade cellulose in this project.

1. 1.Alk lkaline Cell llulase

Co Conclusion

2.

• 2. Mdfa Antiporter

4.c 4.ccdB su suicides sy syst stem 3. 3.Kil il pro rotein

• Mdfa helping tolerate the

high pH conditions in a range from pH 9.0 to PH 9.5.

• Safety precaution
• The E.coil is under effective

control by the ccdB system.

• Kil can enhance

the secretion of Alkaline Cellulase .

Dream Factory

Alkaline Cellulase

Th The ap application of f the “Dream fa factory”

1.We marked on the same piece of paper with water ink before Deinking Experiment. 2.We cut the paper and put them in the control (bacteria liquid without Alkaline cellulase)and fermentation liquid of the "Dream factory”. Deinking Te Test

Dream Factory

Alkaline Cellulase It works!

Experiment result showed that the "Alkaline cellulase dream factory "can effectively promote the deinking efficiency for waste paper.

Human Practice & Our Achivements

PART 4：

Yo Yongyi WANG

Alkaline Cellulase

Dream Factory

 Policy and practice  Our achievements Acknowledgement

Dream Factory

Alkaline Cellulase

• Propagandizing igem in Cultural Festival
• iGEM High School Meetup in Hongkong
• The Communication with SUSTC
• Vis

isiting Paper Mill

• The questionaire

Policy and practice

Dream Factory

Alkaline Cellulase

Propagandizing iGEM in Cultural Festival

• n campus

A happy and successful propaganda...

interacted participants with games

Dream Factory

Alkaline Cellulase

iGEM High School Meetup in Hongkong

always excited...

conducted acadamic exchanges

Dream Factory

Alkaline Cellulase

The Communication with SUSTC-iGEM

Group photo of SZU & SUSTC iGEMers

Communicating...

Dream Factory

Alkaline Cellulase

Visiting private Paper Mill

The most meaningful tour

• ut of recognition

Al2(SO4)3

always curious...

practical apllication

2014/11/2

Our questionnaire

The recognition of Alkaline Cellulase in application The attitudes towards our project replied in industrial production

Dream Factory

Alkaline Cellulase

2014/11/2

Our achievements:

Help to ETH Zurich

ETH Zurich invited us to participate in their survey related to complexity. As feedback, ETH Zurich gave us a badge.

Collabrated with iGEM teams:

senior high school iGEM meetup and SUSTC-iGEM

Improved an exsiting part:

BBa_K1350007 Cel5A, endoglucanase（BBa_K805011 Improved）

Our biobricks:

http://2014.igem.org/Team:SZU-China

2014/11/2

Acknowledgement

Instructor

• Dr. Weizhao Chen

Instructor

• Dr. Jiangxin Wang

Instructor

• Dr. Shengli Tian

2014/11/2

Th Thanks anks ~~~ ~~~