Foundation New approach Practice

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3 Foundation New approach Practice

4 Background Alkanes High energy density Compatible with current infrastructure Project Drop-in replacement of current fuels 4

5 Background Alkane biosynthesis pathway Sensing alkanes Limits of current methods require high-tech equipment need extraction could not detect real-time in vivo condition

6 Background Our solution inconspicuous alkanes chromophoric fluorescent...

7 How to transfer the rough idea to a specific design?

8 Design 1.Inspiration AlkR PalkM AlkM AlkR Alkane Alkane hydroxylase The regulation mechanism of alkane degration in Acinetobacter sp. ADP1

9 Design 2. First Design AlkR PalkM AlkM psb1c3 AlkR Pc AlkR PalkM RFP RFP RFP Alkane RFP RFP RFP

10 What is the cause of the leakage? How to decrease it?

11 Design 3.Mathematical Analysis Reactions Kinetic equations V ga, AlkR AlkR k da, aa 1 2 ' K 2 2 AlkR Pm Pm'' K Alkane AlkR Pm Pm Pm RNAP Pm RNAP k ' ' ' Reporter Pm RNAP Pm RNAP k '' '' '' Reporter Leakage [ Rp] 0 k ' P [ Pm ] k K 0 t 2 2 d 1/ [ AlkR] K 2

12 Output: concentration of RFP Design 3.Mathematical Analysis Excessive ALKR Dynamic Range [ALKR] Leakage Input: concentration of alkane

13 Design 4.Improvment of Construction Change the promoter of ALKR. ALKR B1006 PalkM RFP B1006 psb1c3

14 Design 4.Improvment of Construction Change the promoter of ALKR. Switch the position of ALKR and PalkM-RFP Replace B1006 with a stronger terminator, B0015 ALKR B1006 B0015 PalkM RFP B1006 B0015 psb1c3

15 Design Original Reconstructed The leakage is decreased significantly!

16 Design Leakage 50% Dynamic range 500% We successfully improved AlkSensor s performance

17 Can the optimized alkane sensor distinguish different in vivo input?

18 Experiments and Results In vivo detection of alkanes

19 Experiments and Results In vivo detection of alkanes AlkSensor AlkSensor AlkSensor Strong Alk-producer Weak Alk-producer Null

20 Experiments and Results In vivo detection of alkanes AlkSensor function normally in the in vivo test

21 How to get the quantitative output-input relation of AlkSensor?

22 Experiments and Results Characterization of AlkSensor

23 Experiments and Results Characterization of AlkSensor Add inducer in full dose. Inducers: Hexadecane (C16), tetradecane (C14), Dodecane (C12), Octane (C8) Control group: Water H 2 O

24 Experiments and Results Characterization of AlkSensor Octane (C8) Working curve Pentadecane (C15) Experiment condition: M9 Medium+DMSO AlkSensor s responses to both C8 and C15 are monotone increasing. 0~100nM is the sensitive range of input.

25 A bright future for AlkSensor.

26 Future Work Application 1:Productivity Selection Tc: tetracycline

27 Future Work Application 1:Productivity Selection Higher productivity of alkane faster cell growth rate

28 Future Work Preliminary test of productivity selection Alkane producing module + Productivity Selection module Null + Productivity Selection module The productivity selection strategy work successfully in the preliminary test!

29 Future Work Application 2:Alkane Kit Fuel Exploitation Environmental monitoring

30 Future Work Application 2: Alkane Kit Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Get the sample Add surfactants (triton, DMSO) Cell induction Real-time measurement output Find corresponding alkane concentration Calculate the amount of alkanes in the sample

31 Aim:in vivo, real-time detection of alkanes Alkane Kit Alksensor In vivo Qualitative test Productivity Selection In vitro characterization

32 BioBricks Our Favorite Parts Parts Sandbox Name Type Description Designer Length BBa_K BBa_K Composite Composite Alkane Marker--Device for detection of various alkanes Alkane Selector for intracellular alkane production Boxuan Zeng Yue Liu Boxuan Zeng Yue Liu 2146bp 2662bp Name Type Description Designer Length BBa_K Reporter PalkM promoter with reporter gene Boxuan Zeng Yue Liu 1013bp BBa_K Coding alkane regulatory protein ALKR Boxuan Zeng 927bp BBa_K Reporter AlkR -PSB1C3-Low constitutive Yue Liu 1125bp BBa_K Composite Biosensor for alkane detection Yue Liu Su Wang 2009bp BBa_K Coding YbhGFSR Boxuan Zeng Yang Wu 4971bp BBa_K Composite AlkR -PSB1C3-High constitutive Yue Liu 1125bp BBa_K Composite alkane producing module Boxuan Zeng Yue Liu 1809bp BBa_K Composite YbhGFSR operon Boxuan Zeng 5034bp BBa_K Coding TetA gene under the control of PalkM Boxuan Zeng Shoujie Sun 1529bp BBa_K Composite High expressed Alkane Selector for intracellular alkane production Boxuan Zeng Qingyi Deng 2662bp

33 BioBricks Alkane Marker for detection of various alkanes Alkane Selector for intracellular alkane production

34 Start a new team Human Practice

35 Start a new team

36 Start a new team 1. Foundation igem handbook background of the competition experimental guidance interview of team leaders description of former champions

37 Start a new team 1. Foundation Network Promotion :

38 Start a new team 2. New Approach-- 60 second igem Audio 60 Second igem

39 Start a new team 2. New Approach Mobile Game

40 Start a new team 3. Practice -- Lecture Better synthetic biology, better future

41 Start a new team 3. Practice -- Experiment Guidance

42 Team leader: Sun Min The Chairman of Nankai science asssociation

43 Collaboration BIT-China Debugging & Characterizing RNA thermometer in BBa_K Observed the expression of RFP at different temperatures

44 Collaboration 1.Society institution PetroChina Dagang Petrochemical Company Tainjin Institute of Industrial Biotechnology, Chinese academy of science

45 Communication 2.Universities

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47 Summary Outreach Activities Achievement Foundation IGEM handbook words handbook Contains background, basic knowledge of igem interview and project intriduction New approaches Practice Model igem Jamboree Collaboration & Communication 60 second igem audio Mobile game competition Network Promotion Lectures Experiential guidance Mutually inspired Universities Society Institutions 40 + audios followers Indirectly influenced individuals Collaborate with Dr. Zhou Jun Nankai University starts a new team 12 + team members Ruijing High school is willing to start a team in the future Share ideas with PKU, Fudan, NJU, BIT, OUC BIT, Tsinghua, OCU,PKU Chinese Academy of Science Petro China Petrochemical Company

48 Acknowledgment Special thanks to Instructor Professor Yingjin Yuan Supported by Instructor Doctor Bingzhi Li The Key Laboratory of Systems Bioengineering Tianjin University Jiankun Foundation Tianjin University Visiting Instructor Doctor Patrick Cai Acknowledgment

49 Thank you!