Artificial Organelle Reconstructing the magnetosome membrane in - PowerPoint PPT Presentation

First Step Towards Artificial Organelle —— Reconstructing the magnetosome membrane in E.coli OUC-China 2013

Oce ceanf nfloa loat and nd Oce ceanf nfeel —— OUC-IGEM 2012 Last year…we focused on detection…

OUC-CHINA 2013 This year… We made a Membranous Organelle in prokaryote! What can it do ? How did we make it ? ……

Background Why membrane-bound organelles? Inside the membrane: On the membrane: Isolated Reaction Site Environment

Outline 1. Project part1: Artificial organelle part2: RNA Guardian 2. Future work 3. Judging criteria 4. Human practice 5. Acknowledgements

Analysis What’s the compartment that we want?  Lipid  Enough space  Anchor protein  Easy to use  Compatible

Analysis Only Eukaryotes? Can intracellular membrane exist in prokaryotes ?

Analysis The answer may lie in this species: Magnetospirillum magneticum

Analysis Natural intracellular membrane 3D organization of magnetosomes An ECT reconstruction of Magnetospirillum magneticum sp.AMB-1. Image courtesy of Zhuo Li & Grant Jensen

Analysis The magnetosome membrane  Lipid  Enough space  Anchor enzymes  Easy to use  Compatible ?……

Intracellular Compartment Bacteria Culturing Gene Clustering Compartment Detection

Bacteria Culturing Magnetospirillum Magneticum Microearophilic bacteria---How to culture Contain magnetism---How to detect

Magnetism Detection

Detection Technical Note Channel 1: bacterial through. diameter:40um;length:2000um Channel 2: bacterial sorting under magnetic field. width:2000um;length:3000um Channel 3: bacterial collection and counting. diameter:40um;length:2000um The schematic diagram of our Microfluidic chip

Detection Count

Models of Magnetic Analysis Projection using random function to simulate the movement process inside the microfluidic chip introducing a magnetism detection coefficient to quantify this ability

Models of Magnetic Analysis Simulate the movement process inside the microfluidic chip

Detection Final position with/without magnetic field Dynamic acceleration caused by magnetic field Drag acceeleration caused by microfluidic chip

Models of Magnetic Analysis introducing a magnetism detection coefficient to quantify this ability the density distribution of the bacteria in magnetic field is approxi- mately a piecewise linear function.

Experimental results Magnetospirillum Magneticum AMB-1

Experimental results The E.coli

Gene Clustering Fuction of Magnetosome Island About R5 Region Artifical Gene cluster

Gene Clustering Organization of Magnetosome Island in M.magneticum AMB-1 Image courtesy of xuzheng et.

Gene Clustering Repeats Gene Fragments Regular Gene Sequence So, transform only necessary genes in E.coli Too Large to Transform

Gene Clustering  MamK : Lead invagination derived; Assembly compartment into chain ;  MamI , L, B, Q: Stabilize the compartment chain . Model for magnetosome formation

Gene Clustering The design of artificial magnetosome gene cluster.

Compartment Detection

Compartment Detection MamC::GFP fusion protein MamC is the mostly expressed anchor protein GFP, a widely used reporter protein among all the MMB associated proteins.

Compartment Detection mamC::GFP Compartments ! Laser confocal microscopy result of Control & experimental group mamC::GFP & Artificial Gene Cluster

RNA Guardian

RNA Guardian Introduction Energy Wasting! Energy-efficient! Higher Promoter RBS copy plasmid? 31

RNA Guardian Introduction Translating Not translating Degradation

RNA Guardian Introduction Stabilizing a mRNA using a ribosome?

RNA Guardian Design  Structure of RNA guardian A . Structure of part K1059003 B . Structure of part K1059004

RNA Guardian Design Mode pattern of RNA guardian.

RNA Guardian Design Circuits 3 ’ 5 ’ promoter GFP with lva tag Experiment 1 5 ’ GFP with lva reporter tag Experiment 2 GFP with lva tag 5 ’ -end 5 ’ mRANAguardran 3 ’ 3 ’ -end 3 ’ mRANAg uardran GFP with lva tag Experiment 3 RBS terminater 5 ’ 5 ’ Experiment 4 GFP with lva tag

RNA Guardian Design Comparison of the experimental and control groups by RFU Comparison by RFU Relative increasing

RNA Guardian Result Prediction with modeling Is our design feasible?

RNA Guardian Modeling RBS Calculator

RNA Guardian Modeling RBS Calculator The Control & Experimental Group 3(only plot the RNA) The Control Group The Experimental Group 3

RNA Guardian Modeling Simulated result for control & experimental group The Control Group Experimental Group 4

RNA Guardian Modeling Error Detection and Estimation

RNA Guardian Modeling Result The Stability of mRNA is sorted as: (RBS0 + CDS + RBS1)(+) > (RBS1 + RBS0 + CDs)(+) > (RBS0 + CDs ) (RBS0 + CDS + RBS1)(+):60% relatively increase theoretically (RBS1 + RBS0 + CDs)(+):20% relatively increase theoretically

Future Transmission electron microscopy 1.Transmission Electron Microscopy(TEM) 2.RT-PCR & Catalysis mechanism 3.Make intracellular reactor for health and environment

Part submitted Name Type Description Designer -?- W BBa_K1059003 Regulatory Its transcript can prevent the mRNA itself from being degraded by RNaseE. Qiu Wang Its transcript can prevent the mRNA itself from being degraded by W BBa_K1059004 Regulatory Qiu Wang exonuclease. W BBa_K1059010 Coding RBS J23106+mamI coding squence Wenjun Wang Coding RBS B0032+mamB coding squence Wenjun Wang BBa_K1059013 W BBa_K1059066 Composite GFP-LVA under RNA guardian control Yu Wang Coding mamB coding sequence Wenjun Wang BBa_K1059091 Composite GFP-LVA under J23101 control Yu Wang BBa_K1059001 W BBa_K1059002 Composite GFP-LVA under B0035 control Yu Wang Regulatory DNA segment whose transcript can prevent mRNA degradation by RNaseE. Xue Sun BBa_K1059005 DNA segment whose transcript can prevent mRNA degradation by RNaseE in Regulatory Xue Sun BBa_K1059006 two state . Coding RBS J23106+mamL coding squence Wenjun Wang BBa_K1059011 Coding RBS B0032+mamQ coding squence Wenjun Wang BBa_K1059012 Coding RBS B0032+mamK coding squence Wenjun Wang BBa_K1059014 Coding Promoter J23106 RBS B0032+mamK coding squence Wenjun Wang BBa_K1059015 Coding mamL coding squence Wenjun Wang BBa_K1059017 W BBa_K1059027 Composite GFP-LVA under RNA guardian controld by exonuclease. Qiu Wang Composite GFP-LVA under RNA guardian control Yu Wang BBa_K1059099

Achievement & judging criteria We deserve a Gold medal! 1. Submit a series of new standard Biobrick part and device to MIT. 2. Design RNA guardian device and prove it works well. 3. Improve a Biobrick BBa_K590015 . 4. Utilize microfluidic chip 5. Build the mathematical model reflecting for magnetic detection. 6.Design an approach to analyze the magnetic of bacteria quantitatively. 7.Help Tsinghua University 8.Run lots of human practice, sharing, thinking,discussing and practicing.

Camps & Classes Camp for high school Brainstorming Winter Camp Mini-jamboree of Science and Technology Camp

Visits Between iGEM Teams Visit From SCAU Visit to Tianjin Model iGEM in Peking

Acknowledgement Organizations: Ocean University of China Qingdao Institute of BioEnergy Tianjian University Peking University Bioprocess Technology, Chinese Academy of Sciences Provincial Engineering Laboratory For Biomass Conversion And Process Integration France-China Bio-Mineralization and Nano-Structure Laboratory(Biomnsl) Institute of Oceanology, Chinese Academy of Sciences Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences

Acknowledgement Instructors: Advisors: Xiao-hua Zhang Xu Jian Yang Liu Longfei Wu Guanpin Yang Wei Liu Tian Xiao Li Kang Xianghong Wang Yong Peng Yunxiang Mao Wenjie Wu Chenguang Liu Peiran Zhang Wen Dong Jiaheng Li Zhenmin Bao Weihong Lai Shugang Dong Tianhe Wang Zhihong Tang Jie Yu

Thank you! New Artificial Organelle in prokaryotes