2013 OUC-IGEM 亚洲赛区 演讲PPT，PengYong后期修改。

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

2. Last year…we focused on detection…
Oceanfloat and Oceanfeel
——OUC-IGEM 2012

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

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

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

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

7. Analysis
Only Eukaryotes?
Can intracellular membrane exist in prokaryotes?

8. Analysis
The answer may lie in this species:
Magnetospirillum magneticum

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

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

11. Intracellular Compartment
Bacteria Culturing
Gene Clustering
Compartment Detection

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

13. Magnetism Detection

14. 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

15. Count
Detection

16. 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

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

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

19. 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.
Models of Magnetic Analysis

20. Magnetospirillum
Magneticum
AMB-1
Experimental results

21. The E.coli
Experimental results

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

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

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

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

26. The design of artificial magnetosome gene cluster.
Gene Clustering

27. Compartment Detection

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

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

30. RNA Guardian

31. Energy Wasting!
31
Energy-efficient!
Higher
copy
plasmid?
RBSPromoter
RNA Guardian Introduction

32. Degradation
Translating
Not translating
RNA Guardian Introduction

33. Stabilizing a mRNA
using a ribosome?
RNA Guardian Introduction

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

35. RNA Guardian Design
Mode pattern of RNA guardian.

36. 3’
5’
5’ 5’
3’
5’
GFP with lva tag
GFP with lva tag
GFP with lva tag
GFP with lva tag
Circuits
promoter
GFP with lva
tag
reporter
5’
5’-end
mRANAguardran
3’
3’-end
mRANAguardran
RBS
terminater
Experiment 1
Experiment 4
Experiment 3
Experiment 2
RNA Guardian Design

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

38. Is our design feasible?Prediction with modeling
RNA Guardian Result

39. RNA Guardian Modeling RBS Calculator

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

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

42. Error Detection and Estimation
RNA Guardian Modeling

43. 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

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

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

46. 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.

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

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

49. 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

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

51. Thank you!
New Artificial Organelle in
prokaryotes