ouc-china - 2014.igem.org2014.igem.org/files/presentation/ouc-china_championship.pdfmini plasmid ouc...
TRANSCRIPT
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OUC-China
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lasmid dventurer
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OUTLINE
ProjectOverview
Conjugation
Transfection
Model
Policy & Practice
1
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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
Transfection3
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Transfer exogenous geneticmaterial 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 OriTsequence.
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 ofthe 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 11
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Electrophoretogram DNA Sequencing
Result Conjugation
3000bp
5000bp
1500bp
2000bp
750bp1000bp
500bp
250bp
100bp
This image is from Wikipedia
Mini plasmid OUC
12
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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 14
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Result Conjugation
15
Not finished yet.
But just a matter of time.
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Produce TAT-H4 Lysis Transfection
Transfection
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The function of TAT
Histone H4The structure of nucleosome
TAT-PTD
Design on Fusion Protein Transfection
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Complete the Mini Plasmid Transfection
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3000bp5000bp
1500bp2000bp
750bp1000bp
500bp
250bp100bp
TAT::H4 Transfection
The protein can bind plasmids
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TAT:H4/DNAmass ratio
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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
3000bp5000bp
1500bp2000bp
750bp1000bp
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
22
OD:5mm2
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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 ofL-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
of data
ODE
modelPromotion
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
tetRPR0040
tetR
aTc
lysis
Mechanism
Using aTc to Induce Lysis Modeling
ODE
𝑑 𝑡𝑒𝑡𝑅𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑑𝑡
= 𝐾11
𝑎𝑇𝑐− 𝛼1 𝑡𝑒𝑡𝑅𝑝𝑟𝑜𝑡𝑒𝑖𝑛 + 𝛼2
𝑑 𝑙𝑦𝑠𝑖𝑠
𝑑𝑡= 𝐾2
1
𝑡𝑒𝑡𝑅𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑛 + 𝛽 𝑎𝑇𝑐
28
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Result Modeling
𝑡𝑒𝑡𝑅𝑝𝑟𝑜𝑡𝑒𝑖𝑛 =𝐾1 + 𝛼1 𝑎𝑇𝑐 + 𝐶1𝑒
−𝛼2𝑡
𝛼2 𝑎𝑇𝑐
𝑙𝑦𝑠𝑖𝑠 = 𝐶2 +𝑡 𝐾2 + 𝛽 𝑎𝑇𝑐 𝑡𝑒𝑡𝑅𝑝𝑟𝑜𝑡𝑒𝑖𝑛
𝑛
𝑡𝑒𝑡𝑅𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑛
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The aTc additive amount
The amount of
lysed cells
Estimate
Control
Result Modeling
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Input
Gene
Protein
LysisTopological structure
ODE
Application Modeling
𝑑 𝑝𝑟𝑜𝑡𝑒𝑖𝑛
𝑑𝑡= 𝐾1
𝑙
𝑖𝑛𝑝𝑢𝑡− 𝛼1 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 + 𝛼2
𝑑 𝑙𝑦𝑠𝑖𝑠
𝑑𝑡= 𝐾2
𝑙
𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑛 + 𝛽 𝑖𝑛𝑝𝑢𝑡
30
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ODE
Self-lysis Device Modeling
Self-lysis Regulated by Quorum Sensing
𝑑 𝑐
𝑑𝑡=
𝛽3 𝑖𝑛𝑝𝑢𝑡 𝑛
𝐾1𝑛 + 𝑖𝑛𝑝𝑢𝑡 𝑛
− 𝛼3 𝑐
𝑑 𝑙
𝑑𝑡=
𝛽1𝐾𝑐1𝑚
𝐾𝑐1𝑚 + 𝑐 𝑚
+𝛽2 𝑙𝑢𝑥 𝑝
𝐾𝑐𝑝 + 𝑙𝑢𝑥 𝑝
− 𝛼1 𝑙
𝑑 𝑙𝑢𝑥
𝑑𝑡=
𝛽3𝑘𝑐2𝑞
𝑘𝑐2𝑞 + 𝑐 𝑞
− 𝛼2 𝑙𝑢𝑥
31
pH
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pH=8 pH=5
Diagrams of [c] [lux] and [l]
Result Modeling
𝑚 = 4, 𝑛 = 4, 𝑝 = 4, 𝑞 = 4α1 = 2, α2 = 2, α3 = 2β1 = 2, β2 = 2, β3 = 2
𝑘1 = 1, 𝑘𝑐 = 1, 𝑘𝑐1 = 1, 𝑘𝑐2 = 1
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Simplifying the RP4 plasmid
Self-lysis device automatically
Application in Oral DNA vaccine for fish
Future
33
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Communication
Camp
Outreach
Policy & Practice
34
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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
35
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The mini jamboree Outward bound
Ecology practice Ecology experiment
Camp & Class & Lecture Policy & Practice
The Fourth Science and Technology camp
36
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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
37
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Outreach Policy & Practice
DNA vaccine
coated pellet
Fish feed
Use feed as the carrier of DNA vaccine
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✓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 MaoDr. Xianghong WangProf. Zhenmin BaoProf. Min Wang Prof. Chenguang LiuProf. Zhigang QiuDr. Xiaolong WangProf. Huarong Guo College of Marine Life Science,
Ocean University of China
Acknowledgments
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END