Artificial Organelle Reconstructing the magnetosome membrane in - - PowerPoint PPT Presentation

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Artificial Organelle Reconstructing the magnetosome membrane in - - PowerPoint PPT Presentation

Jun 06, 2023 227 likes •746 views

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 yearwe focused on detection OUC-CHINA 2013 This

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OUC-China 2013 First Step Towards

——Reconstructing the magnetosome membrane in E.coli

Artificial Organelle

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Last year…we focused on detection…

Oce ceanf nfloa loat and nd Oce ceanf nfeel

——OUC-IGEM 2012

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OUC-CHINA 2013

This year…

We made a Membranous Organelle in prokaryote!

What can it do ? How did we make it ? ……

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Background

Why membrane-bound organelles?

Inside the membrane:

Isolated Environment

On the membrane:

Reaction Site

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Outline

1. Project

part1: Artificial organelle part2: RNA Guardian

2. Future work 3. Judging criteria 4. Human practice 5. Acknowledgements

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Analysis

What’s the compartment that we want?

  • Lipid
  • Enough space
  • Anchor protein
  • Easy to use
  • Compatible
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Analysis

Only Eukaryotes?

Can intracellular membrane exist in prokaryotes?

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Analysis

The answer may lie in this species:

Magnetospirillum magneticum

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Analysis

Natural intracellular membrane 3D organization of magnetosomes

An ECT reconstruction of Magnetospirillum magneticum sp.AMB-1. Image courtesy of Zhuo Li & Grant Jensen

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Analysis

The magnetosome membrane

  • Lipid
  • Enough space
  • Anchor enzymes
  • Easy to use
  • Compatible?……
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Intracellular Compartment

Bacteria Culturing Gene Clustering Compartment Detection

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Bacteria Culturing Magnetospirillum Magneticum

Microearophilic bacteria---How to culture Contain magnetism---How to detect

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Magnetism Detection

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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

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Count

Detection

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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

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Simulate the movement process inside the microfluidic chip

Models of Magnetic Analysis

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Detection

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

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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

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Magnetospirillum Magneticum AMB-1

Experimental results

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The E.coli

Experimental results

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Gene Clustering

Fuction of Magnetosome Island About R5 Region Artifical Gene cluster

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Organization of Magnetosome Island

in M.magneticum AMB-1 Image courtesy of xuzheng et.

Gene Clustering

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

Gene Clustering

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  • MamK:

Lead invagination derived; Assembly compartment into chain;

  • MamI, L, B, Q:

Stabilize the compartment chain.

Model for magnetosome formation

Gene Clustering

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The design of artificial magnetosome gene cluster.

Gene Clustering

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Compartment Detection

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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

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mamC::GFP

Compartments!

mamC::GFP & Artificial Gene Cluster

Laser confocal microscopy result of Control & experimental group

Compartment Detection

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RNA Guardian

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Energy Wasting!

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Energy-efficient!

Higher copy plasmid?

RBS Promoter

RNA Guardian Introduction

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Degradation

Translating Not translating

RNA Guardian Introduction

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Stabilizing a mRNA using a ribosome?

RNA Guardian Introduction

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RNA Guardian Design

  • Structure of RNA guardian

A . Structure of part K1059003 B . Structure of part K1059004

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RNA Guardian Design

Mode pattern of RNA guardian.

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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

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Comparison of the experimental and control groups by RFU

Comparison by RFU Relative increasing

RNA Guardian Design

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Is our design feasible? Prediction with modeling

RNA Guardian Result

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RNA Guardian Modeling RBS Calculator

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The Control & Experimental Group 3(only plot the RNA) The Control Group The Experimental Group 3

RNA Guardian Modeling RBS Calculator

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

RNA Guardian Modeling

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Error Detection and Estimation

RNA Guardian Modeling

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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

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Future

Transmission electron microscopy

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

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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

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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.

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Camps & Classes

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

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Visits Between iGEM Teams

Visit From SCAU Visit to Tianjin Model iGEM in Peking

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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

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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

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Thank you!

New Artificial Organelle in prokaryotes