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

Alkaline Cellulase Dream Factory CONTENTS ： Background 1 Project & Design 2 Results 3 Human Practice 4

Alkaline Cellulase Dream Factory PART 1 ： BACKGROUND Zh Zhipeng QI QIU  Paper recycling ； Deinking  Environment and Sustainable Development

Alkaline Cellulase Dream Factory Chinese economy is developing rapidly However, our environment is deteriorating…… The polluted river in Rural China can be seen everywhere!

Alkaline Cellulase Dream Factory Waste paper recycling industry Use toxic chemicals during the deinking Pour the waste water Into the river di directly! Simple and crude equipment

Alkaline Cellulase Dream Factory T he rise of synthetic biology provides a new way to solve this type of environmental question. W e 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.

Alkaline Cellulase Dream Factory PART 1 ： Project & Design Yiti Yiting LU LUO  Four systems of the Dream factory

Alkaline Cellulase D r e a m F a c t o r y Alkaline Cellulase -for the release of ink from the fiber surface by partial hydrolysis of carbohydrate molecules Mdfa Antiporter - resistance to alkaline wastewater Kil - transportation system, enhance the secretion of alkaline cellulase CCDB -Suicide system

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

Alkaline Cellulase Dream Factory Alkaline Cellulase e ndo-(1,4)- β -D-glucanase (EC 3.2.1.4) -------deinking cellulose ink Release of ink from the fiber surface by Alkaline Cellulase partial hydrolysis of carbohydrate molecules.

Alkaline Cellulase D r e a m F a c t o r y Mdfa Antiporter Alterations of cell membrane OH OH OH OH - - - - OH OH OH OH Remodeling of - - - - metabolic patterns OH OH - OH OH - - - OH OH OH OH - - - - Normal Alkaline tolerance bacteria bacteria Active inward transporters of protons

Alkaline Cellulase D r e a m F a c t o r y Mdfa Antiporter Couple the inward movement of protons to the outward of Na+. Maintenance of a stable cytoplasmic pH under conditions of alkaline stress. 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.

Alkaline Cellulase D r e a m F a c t o r y Kil gene 1 Increase the permeability of cell membrane For secretion of alkaline cellulase 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)

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

Alkaline Cellulase Dream Factory Integration FOUR PARTS Alkaline line Cellulas lulase e Dream m Factory ory

Alkaline Cellulase Dream Factory PART 3 ： Results Hu Huan Wei  Four systems of the Dream factory  Deinking Test

Alkaline Cellulase Dream Factory Our Project has four parts ： Alkaline Mdfa Cellulase Antiporter ccdB Suicide Kil Protein System

Alkaline Cellulase Dream Factory Alkaline Cellulase 1.use two methods to detect cellulase activity. Figure 1. Using CMC-Na DNS method Figure 2. transparent zones produced for detection of Alkaline Cellulase by E.coli on CMC-containing solid plate Cellulase can degrade cellulose .

Alkaline Cellulase Dream Factory Alkaline Cellulase 2.The optimum reaction pH and pH stability 3.Research on temperature stability pH=9.0 of Alkaline Cellulase ◇ containing 5mM CaCl 2 ■ without CaCl 2 optimum temperature is 50 ℃ , Optimum reaction pH is 9.0. Ca 2+ can improve stability. Alkaline conditions with strong stability.

Alkaline Cellulase Dream Factory Mdfa Antiporter Growth contrast in LB plate medium with different pH. Mdfa ： promoter+RBS+Mdfa control ： promoter+RBS

Alkaline Cellulase Dream Factory 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

Alkaline Cellulase Dream Factory kil protein 1 0.042 0.045 0.04 0.035 OD 540 VALUE 0.03 0.025 0.018 0.02 0.015 0.01 0.005 0 0 LB PLATE WITHOUT WITH KIL with kil without kil KIL Culture two days later OD 540 value by DNS method measuring Determination of extracellular enzyme activity . Alkaline Cellulase to test kil function Kil can enhance the secretion of alkaline cellulase . 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)

Alkaline Cellulase Dream Factory CCDB suicide system Ou Our E. . co coli can‘t grow in the LB plate medium with IPTG

Alkaline Cellulase Dream Factory 1. 1.Alk lkaline Cell llulase 2. 2. Mdfa Antiporter • Cellulase can successfully • Mdfa helping tolerate the degrade cellulose in this project. high pH conditions in a range from pH 9.0 to PH 9.5. Co Conclusion 4.c 4.ccdB su suicides sy syst stem 3.Kil 3. il pro rotein • Kil can enhance • Safety precaution the secretion of Alkaline • The E.coil is under effective Cellulase . control by the ccdB system.

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

Alkaline Cellulase Dream Factory It works! Experiment result showed that the "Alkaline cellulase dream factory "can effectively promote the deinking efficiency for waste paper.

Alkaline Cellulase Dream Factory PART 4 ： Human Practice & Our Achivements Yo Yongyi WANG  Policy and practice  Our achievements  Acknowledgement

Alkaline Cellulase Dream Factory Policy and practice • Propagandizing igem in Cultural Festival • iGEM High School Meetup in Hongkong • The Communication with SUSTC • Vis isiting Paper Mill • The questionaire

Alkaline Cellulase Dream Factory Propagandizing iGEM in Cultural Festival on campus interacted participants with games A happy and successful propaganda...

Alkaline Cellulase Dream Factory iGEM High School Meetup in Hongkong conducted acadamic exchanges always excited...

Alkaline Cellulase Dream Factory The Communication with SUSTC-iGEM Communicating ... Group photo of SZU & SUSTC iGEMers

Alkaline Cellulase Dream Factory Visiting private Paper Mill Al 2 (SO 4 ) 3 The most meaningful tour out of recognition practical apllication always curious...

Our questionnaire The attitudes towards our project The recognition of Alkaline replied in industrial production Cellulase in application 2014/11/2

Alkaline Cellulase Dream Factory

http://2014.igem.org/Team:SZU-China Our achievements:  Collabrated with iGEM teams: senior high school iGEM meetup and SUSTC-iGEM  Our biobricks:  Improved an exsiting part: BBa_K1350007 Cel5A, endoglucanase （ BBa_K805011 Improved ）  Help to ETH Zurich ETH Zurich invited us to participate in their survey related to complexity. As feedback, ETH Zurich gave us a badge. 2014/11/2

Acknowledgement Instructor Instructor Dr. Jiangxin Wang Dr. Shengli Tian Instructor Dr. Weizhao Chen 2014/11/2

Th Thanks anks ~~~ ~~~ 2014/11/2