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OUC-China 2013
First Step Towards
——Reconstructing the magnetosome membrane in E.coli
Artificial Organelle
Last year…we focused on detection…
Oceanfloat and Oceanfeel——OUC-IGEM 2012
OUC-CHINA 2013This 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:
Isolated Environment
On the membrane:
Reaction Site
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 magnetosomesAn 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:3000umChannel 3:bacterial collection and counting.diameter:40um;length:2000um
The schematic diagram of our
Microfluidic chip
Count
Detection
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
Simulate the movement process inside the microfluidic chip
Models of Magnetic Analysis
Detection
Final position with/without magnetic field
Dynamic acceleration caused by magnetic field
Drag acceeleration caused by microfluidic chip
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
MagnetospirillumMagneticum
AMB-1
Experimental results
The E.coli
Experimental results
Gene Clustering
Fuction of Magnetosome Island
About R5 Region
Artifical Gene cluster
Organization of Magnetosome Islandin M.magneticum AMB-1
Image courtesy of xuzheng et.
Gene Clustering
Repeats Gene Fragments
Regular Gene Sequence
Too Large to Transform
So, transform only necessary genes in E.coli
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.
Gene Clustering
Compartment Detection
Compartment Detection
MamC::GFPfusion protein
MamC is the mostly expressed anchor protein protein among all the MMB associated proteins. GFP, a widely used reporter
mamC::GFP
Compartments!
mamC::GFP&Artificial Gene Cluster
Laser confocal microscopy result of
Control & experimental group
Compartment Detection
RNA Guardian
Energy Wasting!
31
Energy-efficient!
Higher copy
plasmid?RBSPromoter
RNA Guardian Introduction
Degradation
Translating
Not translating
RNA Guardian Introduction
Stabilizing a mRNA
using a ribosome?
RNA Guardian Introduction
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.
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
tagreporter
5’5’-end mRANAguardran
3’3’-end mRANAguardran
RBS
terminater
Experiment 1
Experiment 4
Experiment 3
Experiment 2
RNA Guardian Design
Comparison of the experimental
and control groups by RFU
Comparison by RFU Relative increasing
RNA Guardian Design
Is our design feasible?Prediction with modeling
RNA Guardian Result
RNA Guardian Modeling RBS Calculator
The Control & Experimental Group 3(only plot the RNA)
The Control Group
The Experimental Group 3
RNA Guardian Modeling RBS Calculator
Simulated result for control & experimental group
The Control Group
Experimental Group 4
RNA Guardian Modeling
Error Detection and Estimation
RNA Guardian Modeling
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
W BBa_K1059004 Regulatory Its transcript can prevent the mRNA itself from being degraded byexonuclease. 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 intwo 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
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
Brainstorming Winter Camp Camp for high school
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
Instructors:Xiao-hua ZhangGuanpin Yang
Advisors:Xu JianLongfei WuTian XiaoXianghong WangYunxiang MaoChenguang LiuWen DongZhenmin BaoShugang DongZhihong TangJie Yu
Yang LiuWei LiuLi KangYong PengWenjie WuPeiran ZhangJiaheng LiWeihong LaiTianhe Wang
Acknowledgement
Thank you!
New Artificial Organelle in
prokaryotes
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