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

OUC-China

lasmid dventurer

OUTLINE Project Overview Conjugation Transfection Model Policy & Practice 1

Three Ways of Transfection Lentivirus: the long preparation Lipofectamine: the toxicity Particle bombardment: the high expense 2

Overview A novel model of plasmid transfer Double plasmids system Conjugation Product the fusion protein TAT-H4 Lysis Transfection 3

Conjugation Transfer exogenous genetic Zebrafish 's intestines material by conjugation  Reduce damage  Transfer efficiently 4

RP4 Conjugation Requirements Conjugation  Features a broad conjugation Spectrum  IncP α  The length of this plasmid is 60kb.  Carries many kinds of resistant genes 5

RP4 Conjugation Requirements Conjugation 1. Tra and Trb gene clusters 2.Conjugative transfer origin Tra gene cluster OriT nick site Mating pair formation system(Mpf) RP4 relaxosome nick site 6

Double-plasmid-system Conjugation  Mini plasmid contains function gene and OriT sequence.  Plasmid RP4 create conjugative conditions. 8

Design on Double-plasmid-system Conjugation The double plasmids system 9

Result Conjugation Resistances of the donor and recipient cell Chl R HB101 TOP10 Chloramphenicol Streptomycin (mini plasmid and plasmid RP4 (genome provide) provide) 10

Result Conjugation The experiment results Top10 HB101 HB101 & Top10 Str Chl Str & Chl Culture the donor, recipient, and mixture on selective medium 11

Result Conjugation Electrophoretogram DNA Sequencing 5000bp 3000bp 2000bp 1500bp 1000bp Mini plasmid OUC 750bp 500bp 250bp 100bp This image is from Wikipedia 12

Result Conjugation Conjugation with Vibrio harveyi control group experimental group 13

Result Conjugation OriT of another resistant plasmid Str Chl Str & Chl Top10 HB101 HB101 & Top10 Culture the donor, recipient, and mixture on selective medium 14

Result Conjugation Not finished yet. But just a matter of time. 15

Transfection Lysis Transfection Produce TAT-H4 16

Design on Fusion Protein Transfection TAT-PTD The function of TAT The structure of nucleosome Histone H4 17

Complete the Mini Plasmid Transfection 18

TAT::H4 Transfection The protein can bind plasmids TAT:H4/DNA mass ratio 5000bp 3000bp 2000bp 1500bp 1000bp 750bp 500bp 250bp 100bp 19

TAT::H4 Transfection The protein can protect plasmids 5000bp 3000bp 2000bp 1500bp 1000bp 750bp 500bp 250bp 100bp From Lane 1 to 6. TAT:H4/DNA mass ratio reached 8:1, 6:1, 4:1, 2:1, 1:1, 20 0:1. Lane 7 is pcDNA3.1(+)-EGFP and lane 8 is Marker DL5000

TAT::H4 Transfection The result of Rt-PCR 3000bp 2000bp 1500bp 1000bp 750bp 500bp 250bp 100bp 21

TAT::H4 Transfection OD:5mm 2 The efficiency of transfection of injecting TAT-H4-plasmid is higher 92.41% than injecting plasmid only at least. 22

Design of Lysis Device Transfection Lysis Inducible promoter 23

Design of Lysis Device Transfection 24

Design of Lysis Device Transfection Concentration of Time after induction L-arabinose 3 hours 6 hours 9 hours 1 μ M 0.322 0.745 0.901 3 μ M 0.247 0.741 0.927 6 μ M 0.209 0.733 0.891 10 μ M 0.191 0.750 0.924 100 μ M 0.183 0.721 0.848 10 mM 0.132 0.631 0.685 25

Modeling lysis Quorum L-arabinose aTc sensing Analysis ODE Promotion of data model 26

Using L-arabinose to Induce Lysis Modeling The line chart of the experimental data The device A has worked! 27

Using aTc to Induce Lysis Modeling Mechanism tetR aTc lysis tetR P J23106 P R0040 𝑒 𝑢𝑓𝑢𝑆 𝑞𝑠𝑝𝑢𝑓𝑗𝑜 1 = 𝐿 1 𝑏𝑈𝑑 − 𝛽 1 𝑢𝑓𝑢𝑆 𝑞𝑠𝑝𝑢𝑓𝑗𝑜 + 𝛽 2 𝑒𝑢 ODE 𝑒 𝑚𝑧𝑡𝑗𝑡 1 = 𝐿 2 𝑜 + 𝛾 𝑏𝑈𝑑 𝑒𝑢 𝑢𝑓𝑢𝑆 𝑞𝑠𝑝𝑢𝑓𝑗𝑜 28

Result Modeling 𝑢𝑓𝑢𝑆 𝑞𝑠𝑝𝑢𝑓𝑗𝑜 = 𝐿 1 + 𝛽 1 𝑏𝑈𝑑 + 𝐷 1 𝑓 −𝛽 2 𝑢 𝛽 2 𝑏𝑈𝑑 𝑜 𝑢 𝐿 2 + 𝛾 𝑏𝑈𝑑 𝑢𝑓𝑢𝑆 𝑞𝑠𝑝𝑢𝑓𝑗𝑜 𝑚𝑧𝑡𝑗𝑡 = 𝐷 2 + 𝑜 𝑢𝑓𝑢𝑆 𝑞𝑠𝑝𝑢𝑓𝑗𝑜 29

Result Modeling Estimate The aTc additive The amount of lysed cells amount Control 29

Application Modeling Input Gene Lysis Topological structure Protein 𝑒 𝑞𝑠𝑝𝑢𝑓𝑗𝑜 𝑚 = 𝐿 1 𝑗𝑜𝑞𝑣𝑢 − 𝛽 1 𝑞𝑠𝑝𝑢𝑓𝑗𝑜 + 𝛽 2 𝑒𝑢 ODE 𝑒 𝑚𝑧𝑡𝑗𝑡 𝑚 = 𝐿 2 𝑞𝑠𝑝𝑢𝑓𝑗𝑜 𝑜 + 𝛾 𝑗𝑜𝑞𝑣𝑢 𝑒𝑢 30

Self-lysis Device Modeling Self-lysis Regulated by Quorum Sensing ODE 𝛾 3 𝑗𝑜𝑞𝑣𝑢 𝑜 𝑒 𝑑 𝑒𝑢 = 𝑜 + 𝑗𝑜𝑞𝑣𝑢 𝑜 − 𝛽 3 𝑑 𝐿 1 𝑛 𝛾 2 𝑚𝑣𝑦 𝑞 𝑒 𝑚 𝛾 1 𝐿 𝑑1 𝑒𝑢 = 𝑛 + 𝑑 𝑛 + 𝑞 + 𝑚𝑣𝑦 𝑞 − 𝛽 1 𝑚 𝐿 𝑑1 𝐿 𝑑 pH 𝑟 𝑒 𝑚𝑣𝑦 𝛾 3 𝑙 𝑑2 = 𝑟 + 𝑑 𝑟 − 𝛽 2 𝑚𝑣𝑦 𝑒𝑢 𝑙 𝑑2 31

Result Modeling 𝑛 = 4, 𝑜 = 4, 𝑞 = 4, 𝑟 = 4 α 1 = 2, α 2 = 2, α 3 = 2 Diagrams of [c] [lux] and [l] β 1 = 2, β 2 = 2, β 3 = 2 𝑙 1 = 1, 𝑙 𝑑 = 1, 𝑙 𝑑1 = 1, 𝑙 𝑑2 = 1 pH=8 pH=5 32

Future Simplifying the RP4 plasmid Self-lysis device automatically Application in Oral DNA vaccine for fish 33

Policy & Practice Communication Camp Outreach 34

Communication Policy & Practice Qingdao Institute of Biomass The Central China iGEMers' Energy and Bioprocess Technology Consortium Beijing Normal University iGEM team Peking iGEM team 35

Camp & Class & Lecture Policy & Practice The Fourth Science and Technology camp The mini jamboree Outward bound Ecology experiment 36 Ecology practice

Outreach Policy & Practice The investigation of biological products transport  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 37

Outreach Policy & Practice Use feed as the carrier of DNA vaccine coated Fish DNA vaccine pellet feed

Achievements ✓ 11 BioBricks to part registry ✓ An innovative method to carry exogenous DNA from prokaryote to Eukaryote ✓ Modeling standard for lysis device ✓ Policy and practice 39

Biobricks 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, Wenqi Li 1427 combined with OriTRP4. Used to test plasmid mobility. BBa_K1439002 Composite This part contains a reporter gene BBa_J04450, Wenqi Li 1448 combined with OriTR. Used to test plasmid mobility. BBa_K1439003 Composite [OriTR-RFP]+[lysis] Wenqi Li 3379 BBa_K1439004 Coding TAT-H4 Zhaoliang 393 Chen BBa_K1439005 Coding TAT-PTD Zhaoliang 36 Chen BBa_K1439006� Coding Histone H4 Zhaoliang 312 Chen BBa_K1439007 Regulatory CMV promoter Zhaoliang 588 Chen BBa_K1439008 Composite TAT-H4-B0015 Zhaoliang 530 Chen BBa_K1439009� Device Lysis device induced by L-arabinose Ming Jiang 1923 BBa_K1439010 Device Lysis device induced by aTc Ming Jiang 2738 40

Judging Form ✓ 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. 41

Acknowledgments Instructors Guanpin Yang Xiaohua Zhang 42

Acknowledgments 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 43

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