OUC-China lasmid dventurer OUTLINE Project Overview Conjugation - - PowerPoint PPT Presentation

OUC-China

lasmid dventurer

OUTLINE

Project

Overview Conjugation Transfection

Model Policy & Practice

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Lentivirus: the long preparation Lipofectamine: the toxicity

Particle bombardment: the high expense

Three Ways of Transfection

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Overview

A novel model of plasmid transfer

Double plasmids system Conjugation Product the fusion protein TAT-H4 Lysis Transfection

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Transfer exogenous genetic material by conjugation

• Reduce damage
• Transfer efficiently

Zebrafish's intestines

Conjugation

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• Features a broad

conjugation Spectrum

• IncPα
• The length of this plasmid

is 60kb.

• Carries many kinds of

resistant genes

RP4 Conjugation Requirements

Conjugation

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Tra gene cluster

OriT nick site

• 1. Tra and Trb gene clusters 2.Conjugative transfer origin

Mating pair formation system(Mpf) RP4 relaxosome nick site

RP4 Conjugation Requirements

Conjugation

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• Mini plasmid contains

function gene and OriT sequence.

• Plasmid RP4 create

conjugative conditions.

Double-plasmid-system

Conjugation

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The double plasmids system

Design on Double-plasmid-system

Conjugation

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Resistances of the donor and recipient cell

HB101

Chloramphenicol (mini plasmid and plasmid RP4 provide)

TOP10

Streptomycin (genome provide)

ChlR

Result

Conjugation

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The experiment results

Result

Conjugation

Str Chl Str & Chl HB101 & Top10 Top10 HB101

Culture the donor, recipient, and mixture on selective medium

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Electrophoretogram DNA Sequencing

Result

Conjugation

3000bp 5000bp 1500bp 2000bp 750bp 1000bp 500bp 250bp 100bp

This image is from Wikipedia

Mini plasmid OUC

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Conjugation with Vibrio harveyi

Result

Conjugation

control group experimental group

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Result

Conjugation

Str Chl Str & Chl HB101 & Top10 Top10 HB101

OriT of another resistant plasmid

Culture the donor, recipient, and mixture on selective medium

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Result

Conjugation

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Not finished yet. But just a matter of time.

Produce TAT-H4 Lysis Transfection

Transfection

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The function of TAT Histone H4 The structure of nucleosome TAT-PTD

Design on Fusion Protein

Transfection

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Complete the Mini Plasmid

Transfection

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3000bp 5000bp 1500bp 2000bp 750bp 1000bp 500bp 250bp 100bp

TAT::H4

Transfection

The protein can bind plasmids

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TAT:H4/DNA mass ratio

From Lane 1 to 6. TAT:H4/DNA mass ratio reached 8:1, 6:1, 4:1, 2:1, 1:1, 0:1. Lane 7 is pcDNA3.1(+)-EGFP and lane 8 is Marker DL5000

3000bp 5000bp 1500bp 2000bp 750bp 1000bp 500bp 250bp 100bp

The protein can protect plasmids

TAT::H4

Transfection

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TAT::H4

Transfection

The result of Rt-PCR

3000bp 1500bp 2000bp 750bp 1000bp 500bp 250bp 100bp

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The efficiency of transfection of injecting TAT-H4-plasmid is higher 92.41% than injecting plasmid only at least.

TAT::H4

Transfection

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OD:5mm2

Inducible promoter

Lysis

Design of Lysis Device

Transfection

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Design of Lysis Device

Transfection

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Design of Lysis Device

Transfection

Concentration of L-arabinose Time after induction 3 hours 6 hours 9 hours 1 μM 0.322 0.745 0.901 3 μM 0.247 0.741 0.927 6 μM 0.209 0.733 0.891 10 μM 0.191 0.750 0.924 100 μM 0.183 0.721 0.848 10 mM 0.132 0.631 0.685

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lysis

L-arabinose aTc Quorum sensing Analysis

• f data

ODE model Promotion

Modeling

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The device A has worked!

Using L-arabinose to Induce Lysis

Modeling

The line chart of the experimental data

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PJ23106

tetR

PR0040

tetR

aTc

lysis Mechanism

Using aTc to Induce Lysis

Modeling

ODE

𝑒 𝑢𝑓𝑢𝑆𝑞𝑠𝑝𝑢𝑓𝑗𝑜 𝑒𝑢 = 𝐿1 1 𝑏𝑈𝑑 − 𝛽1 𝑢𝑓𝑢𝑆𝑞𝑠𝑝𝑢𝑓𝑗𝑜 + 𝛽2 𝑒 𝑚𝑧𝑡𝑗𝑡 𝑒𝑢 = 𝐿2 1 𝑢𝑓𝑢𝑆𝑞𝑠𝑝𝑢𝑓𝑗𝑜

𝑜 + 𝛾 𝑏𝑈𝑑

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Result

Modeling

𝑢𝑓𝑢𝑆𝑞𝑠𝑝𝑢𝑓𝑗𝑜 = 𝐿1 + 𝛽1 𝑏𝑈𝑑 + 𝐷1𝑓−𝛽2

𝑢

𝛽2 𝑏𝑈𝑑 𝑚𝑧𝑡𝑗𝑡 = 𝐷2 + 𝑢 𝐿2 + 𝛾 𝑏𝑈𝑑 𝑢𝑓𝑢𝑆𝑞𝑠𝑝𝑢𝑓𝑗𝑜

𝑜

𝑢𝑓𝑢𝑆𝑞𝑠𝑝𝑢𝑓𝑗𝑜

𝑜

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The aTc additive amount

The amount of lysed cells

Estimate Control

Result

Modeling

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Input Gene Protein Lysis

Topological structure ODE

Application

Modeling

𝑒 𝑞𝑠𝑝𝑢𝑓𝑗𝑜 𝑒𝑢 = 𝐿1 𝑚 𝑗𝑜𝑞𝑣𝑢 − 𝛽1 𝑞𝑠𝑝𝑢𝑓𝑗𝑜 + 𝛽2 𝑒 𝑚𝑧𝑡𝑗𝑡 𝑒𝑢 = 𝐿2 𝑚 𝑞𝑠𝑝𝑢𝑓𝑗𝑜 𝑜 + 𝛾 𝑗𝑜𝑞𝑣𝑢

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ODE

Self-lysis Device

Modeling

Self-lysis Regulated by Quorum Sensing

𝑒 𝑑 𝑒𝑢 = 𝛾3 𝑗𝑜𝑞𝑣𝑢 𝑜 𝐿1

𝑜 + 𝑗𝑜𝑞𝑣𝑢 𝑜 − 𝛽3 𝑑

𝑒 𝑚 𝑒𝑢 = 𝛾1𝐿𝑑1

𝑛

𝐿𝑑1

𝑛 + 𝑑 𝑛 +

𝛾2 𝑚𝑣𝑦 𝑞 𝐿𝑑

𝑞 + 𝑚𝑣𝑦 𝑞 − 𝛽1 𝑚

𝑒 𝑚𝑣𝑦 𝑒𝑢 = 𝛾3𝑙𝑑2

𝑟

𝑙𝑑2

𝑟 + 𝑑 𝑟 − 𝛽2 𝑚𝑣𝑦

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pH

pH=8 pH=5

Diagrams of [c] [lux] and [l]

Result

Modeling

𝑛 = 4, 𝑜 = 4, 𝑞 = 4, 𝑟 = 4 α1 = 2, α2 = 2, α3 = 2 β1 = 2, β2 = 2, β3 = 2 𝑙1 = 1, 𝑙𝑑 = 1, 𝑙𝑑1 = 1, 𝑙𝑑2 = 1

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Simplifying the RP4 plasmid Self-lysis device automatically Application in Oral DNA vaccine for fish

Future

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Communication Camp Outreach

Policy & Practice

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Qingdao Institute of Biomass Energy and Bioprocess Technology The Central China iGEMers' Consortium Beijing Normal University iGEM team Peking iGEM team

Communication

Policy & Practice

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The mini jamboree Outward bound

Ecology practice

Ecology experiment

Camp & Class & Lecture

Policy & Practice

The Fourth Science and Technology camp

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 Transport between cities  Parts submission to iGEM officials We have done…

 Investigate the relevant government department and

different express companies.

 Write to the State Post Bureau of China.  Make a proposal to iGEM officials

Outreach

Policy & Practice

The investigation of biological products transport

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Outreach

Policy & Practice

DNA vaccine

coated pellet Fish feed

Use feed as the carrier of DNA vaccine

✓11 BioBricks to part registry ✓An innovative method to carry exogenous DNA from prokaryote to Eukaryote ✓ Modeling standard for lysis device ✓ Policy and practice

Achievements

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Name Type Description Designer Length BBa_K1439000 Conjugation Origin of transfer for the RP4-plasmid nic region. Wenqi Li 350 BBa_K1439001 Composite This part contains a reporter gene BBa_J04450, combined with OriTRP4. Used to test plasmid mobility. Wenqi Li 1427 BBa_K1439002 Composite This part contains a reporter gene BBa_J04450, combined with OriTR. Used to test plasmid mobility. Wenqi Li 1448 BBa_K1439003 Composite [OriTR-RFP]+[lysis] Wenqi Li 3379 BBa_K1439004 Coding TAT-H4 Zhaoliang Chen 393 BBa_K1439005 Coding TAT-PTD Zhaoliang Chen 36 BBa_K1439006 Coding Histone H4 Zhaoliang Chen 312 BBa_K1439007 Regulatory CMV promoter Zhaoliang Chen 588 BBa_K1439008 Composite TAT-H4-B0015 Zhaoliang Chen 530 BBa_K1439009 Device Lysis device induced by L-arabinose Ming Jiang 1923 BBa_K1439010 Device Lysis device induced by aTc Ming Jiang 2738

Biobricks

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✓ Our new BioBrick Parts were designed and they can work as expected. ✓ We characterized our including information and behavior. ✓ We submitted our new part to the iGEM Parts Registry. ✓ We sent two standard biological parts to Peking iGEM team. And we have helped Peking to test the two parts. ✓ During the work time, we did investigations about biological products transport and vaccines about our project.

Judging Form

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Xiaohua Zhang Guanpin Yang

Acknowledgments

Instructors

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• Prof. Yunxiang Mao
• Dr. Xianghong Wang
• Prof. Zhenmin Bao
• Prof. Min Wang
• Prof. Chenguang Liu
• Prof. Zhigang Qiu
• Dr. Xiaolong Wang
• Prof. Huarong Guo

College of Marine Life Science, Ocean University of China

Acknowledgments

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END