OUC-China

lasmid dventurer

OUTLINE

ProjectOverview

Conjugation

Transfection

Model

Policy & Practice

1

Lentivirus: the long preparation

Lipofectamine: the toxicity

Particle bombardment: the high expense

Three Ways of Transfection

2

Overview

A novel model of plasmid transfer

Double plasmids system

Conjugation

Product the fusion protein TAT-H4

Lysis

Transfection3

Transfer exogenous geneticmaterial by conjugation

Reduce damage

Transfer efficiently

Zebrafish's intestines

Conjugation

4

Features a broad conjugation Spectrum

IncPα

The length of this plasmid is 60kb.

Carries many kinds of resistant genes

RP4 Conjugation Requirements Conjugation

5

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

6

Mini plasmid contains function gene and OriTsequence.

Plasmid RP4 create conjugative conditions.

Double-plasmid-system Conjugation

8

The double plasmids system

Design on Double-plasmid-system Conjugation

9

Resistances ofthe donor and recipient cell

HB101

Chloramphenicol(mini plasmid and plasmid RP4

provide)

TOP10

Streptomycin(genome provide)

ChlR

Result Conjugation

10

The experiment results

Result Conjugation

Str

Chl

Str & Chl

HB101 & Top10 Top10 HB101

Culture the donor, recipient, and mixture on selective medium 11

Electrophoretogram DNA Sequencing

Result Conjugation

3000bp

5000bp

1500bp

2000bp

750bp1000bp

500bp

250bp

100bp

This image is from Wikipedia

Mini plasmid OUC

12

Conjugation with Vibrio harveyi

Result Conjugation

control group experimental group

13

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

Result Conjugation

15

Not finished yet.

But just a matter of time.

Produce TAT-H4 Lysis Transfection

Transfection

16

The function of TAT

Histone H4The structure of nucleosome

TAT-PTD

Design on Fusion Protein Transfection

17

Complete the Mini Plasmid Transfection

18

3000bp5000bp

1500bp2000bp

750bp1000bp

500bp

250bp100bp

TAT::H4 Transfection

The protein can bind plasmids

19

TAT:H4/DNAmass 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

3000bp5000bp

1500bp2000bp

750bp1000bp

500bp

250bp

100bp

The protein can protect plasmids

TAT::H4 Transfection

20

TAT::H4 Transfection

The result of Rt-PCR

3000bp

1500bp

2000bp

750bp

1000bp

500bp

250bp

100bp

21

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

Inducible promoter

Lysis

Design of Lysis Device Transfection

23

Design of Lysis Device Transfection

24

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

25

lysis

L-arabinose aTc Quorum

sensing

Analysis

of data

ODE

modelPromotion

Modeling

26

The device A has worked!

Using L-arabinose to Induce Lysis Modeling

The line chart of the experimental data

27

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

Result Modeling

𝑡𝑒𝑡𝑅𝑝𝑟𝑜𝑡𝑒𝑖𝑛 =𝐾1 + 𝛼1 𝑎𝑇𝑐 + 𝐶1𝑒

−𝛼2𝑡

𝛼2 𝑎𝑇𝑐

𝑙𝑦𝑠𝑖𝑠 = 𝐶2 +𝑡 𝐾2 + 𝛽 𝑎𝑇𝑐 𝑡𝑒𝑡𝑅𝑝𝑟𝑜𝑡𝑒𝑖𝑛

𝑛

𝑡𝑒𝑡𝑅𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑛

29

The aTc additive amount

The amount of

lysed cells

Estimate

Control

Result Modeling

29

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

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

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

32

Simplifying the RP4 plasmid

Self-lysis device automatically

Application in Oral DNA vaccine for fish

Future

33

Communication

Camp

Outreach

Policy & Practice

34

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

The mini jamboree Outward bound

Ecology practice Ecology experiment

Camp & Class & Lecture Policy & Practice

The Fourth Science and Technology camp

36

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

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

39

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

40

✓ 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

41

Xiaohua Zhang Guanpin Yang

Acknowledgments

Instructors

42

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

43

END