Powerful Precisely

Background Digital Signal

Background

Oscillation in Synthetic biology

Synthetic biologists’ achievements

1st 2nd

1st 2nd

3rd 4th

Background

MAGIC circuitCoupling among colonies

lux pR

H2O2 ArcAB

Mechanism

luxI aiiA

ndh sfGFP

H2O2

Coupling Reporter

LuxR-AHL

AHL

Oscillator

RBSB0034

Lux pRR0062B0012B0010

luxRC0062

Lux plR0063 Quorum

Sensing

Welcome Conductor Catalase!

catalase

H2O O2

A controllable promoter

Catalase

E. coli

Mechanism

To realize thisof E.coli, we constructed…

Circuit Construction

No. Plasmid Backbone CopyOrigin

Copynumber

A1 RBSB0034

luxI-LVAC0061

RBSB0034B0012B0010 B0012B0010

gfp-LVAJ04031

pSB1C3-gfp-luxI pSB1C3 pMB1High

(100~300)

p3H ColE1Middle(18~22)

A2 RBSB0034

luxI-LVAC0061

RBSB0034B0012B0010 B0012B0010

sfgfp-LVAJ04031

pSB1C3-sfgfp-luxI pSB1C3 pMB1High

(100~300)

p3H ColE1Middle(18~22)

B aiiA-LVAC0060

RBSB0034 B0012B0010

pSB3T5-aiiApSB3T5 p15A Middle

(10~12)

C

ndh-LVARBS

B0034 B0012B0010

pSB4K5-ndh

pSB4K5 pSC101Low(5)

Oscillator: A & B plasmidsNO. Plasmids

A pSB1C3-gfp/sfgfp-luxI

B pSB3T5-aiiA

RBSB0034

luxI-LVAC0061

RBSB0034B0012B0010 B0012B0010

gfp/sfgfp-LVAJ04031

aiiA-LVAC0060

RBSB0034 B0012B0010

Circuit Construction

NO. Parts Submitted

1

2

BBa 1036002

BBa 1036003

Parts UsedNo.

1

2

3

4

BBa_K546000

BBa_I763020

BBa_F2621

BBa_K546001

Coupling: C plasmidNO. Plasmids

C pSB4K5-ndh

NO. Parts Submitted

1 BBa_K1036000

2 BBa_K1036001

NO. Parts Used

3 BBa_K546001

ndh-LVARBS

B0034 B0012B0010

Circuit Construction

Advantage:

High AHL concentration

(A) Swimming Pool Model

Disadvantages:• Hard to control• Increasing RFU baseline

Microfluidic

(B) Passage ModelMicrofluidic

Oscillation in a single colony of BL21(DE3)

Circuit:pSB1C3-gfp-luxI & pSB3T5-aiiA plasmids

(A) Swimming-pool (B) Passage

Results

Host casting

Strain Type Growing Rate Other Reasons we choose it

DH5α K Slow Belong to K strain as MG1655

BL21 (DE3) B Faster

1) Competent in transformation and protein

expression; 2) ndh gene is originally expressed in BL21 (DE3)

BL21 (wild type) B FastestA BL21 strain with less artificial modification

Results

Weakness of DH5α

170130

100

70

55

40

35

25

gfp-aiiA-ndhSP

gfp-aiiASP

pSB1C3-gfpSP

DH5αSP

Ndh AiiA LuxI

P-Precipitate

S-Supernatant

Results

GFP

Oscillation in Microfluidic of BL21(WT)

Time (min)

0 100 200 300 400 500 600

RF

U(a

.u.)

10

20

30

40

50

60

70

80

90Line 1

Line 2

Line 3

High Copy Numbers

(A) pSB1C3-gfp-luxI & pSB3T5-aiiA

Results

Time (min)

0 100 200 300 400 500

RF

U (

a.u

.)

150

200

250

300

350

400

Middle Copy Numbers

(B) p3H-gfp-luxI & pSB3T5-aiiA

Time (min)

0 50 100 150 200 250 300 350

RFU

0

500

1000

1500

2000

2500

3000

3500BL21

p3H-gfp-aiiA/BL21

pSB1C3-gfp-aiiA/BL21

(B)

Oscillation Phenomenon of BL21(WT)Results

Time (min)

0 50 100 150 200 250 300 350

RFU

-100

0

100

200

300

400

500BL21

p3H-gfp-luxI / BL21

pSB1C3-gfp-luxI / BL21

(A)

MircofluidicDevice

LVA-TagCopy number

Ratio

StrainComparison

Complete the comparison of different strains’ effect on oscillation

Confirm most suitable microfluidic parameters for oscillation.

Amplify the effect of different copy numbers

Confirm that every part is functioning as expected (i.e. LVA-tag).

Swimming-pool-like Array

Passage Array

DH5aBL21 (DE3)BL21 (WT)

pMB1/ColE1p15A

pSC101

LVALAA

Accomplishment Future Goal

Figure 1. Delay differential equations (DDE)

Reference: Danino, T., Mondragon-Palomino, O., Tsimring, L., & Hasty, J., A synchronized quorum of genetic clocks. Nature 463, 326-330 (2010)

aiiA

LuxI

InternalAHL

ExternalAHL

Modeling & Software

Four DDEs

What is DDE??

2

2

1

( ( ))

1 ( ( ))

i

i

H t

k H t

DDE is short for delay differential equation

MATLAB

4

0

4

0

[1 ( / ) ] ( , )1 ( )

[1 ( / ) ] ( , )1 ( )

AA

AI

AAC d d P

t f A I

AIC d d P

t f A I

Modeling & Software

Modeling

DDE can perfectly simulate our circuit.

Figure 2. Hill function

Figure 3. The concentration change of the four main proteins

Figure 4. The fluoresence change of each trap

Modeling & Software

Modeling

So, modeling is finished Isn’t it?

NO! Programmer

Computerilliterate

So easy

Help me

OK…I will make a GUI user interface

Modeling & Software

Modeling

Figure 5. The main user interface of Gene OS

Help message is here

Optimaize the experiment conditions

Input boxes

Noisy system

Modeling & Software

Modeling

Software

Figure 6. Gene OS

Gene O Sscillate imulatorModeling &

Software

Software

Modeling

An own-designed

Image process software

Humanized & intelligent

Quick & efficient

ImageMe

Modeling & Software

Software

Modeling

Figure 7. The main user interface of ImageMe

Simpler and

easier.

Modeling & Software

Software

Modeling

Figure 8. One example image of microfluidic

Figure 9. The software output the image after edge processing

Edge Detection

Figure 10. The fluorescence change in the same position

Figure 11. The fluorescence change calculated by ImageMe using gray level integration

Gray Level integrationModeling &

Software

Software

Modeling

Figure 12. The Resultss of test images given by ImageMe

Gene OS ImageMe

Designed only for the experiments and solve the problems we met

Modeling & Software

Software

Modeling

2013/10/5

Applications

MRI

Physical method

Traditional methods

Chemical method

CT

How to combine diagnosis and treatment together ？

Applications

Diagnosis Treatment

Synthetic Biology ？

Cell Metabolism

Oxidant Stress

ROS : reactive oxygen species

Normal CellCancer CellDead Cell

maximum

minimum

Peng Huang et al, Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nature Reviews Drug Discovery Volume 8(2009)

Minimum< ROS Value < Maximum: cancer cellCTApplications

ROS biosensing

maximum

minimum

Advantages

1. Sensitive

2. Robust

•What if we change our GFP to a certain toxalbumin？

Jeff Hasty et al. A sensing array of radically coupled genetic ‘biopixels’. Nature 481, 39-44(2012)

CTApplications

Periodical protein synthesis

May be we can realize periodical toxalbumin synthesis which will be more gentle for cancerous person.

Outreach

1.Lecture2.Campus Fair

3.Collaboration

LectureOutreach

ExhibitionOutreach

GamesOutreach

Bio bangOutreach

fresco in Furong TunnelOutreach

XMU-China

Peking University

NanJin UniversityFrance

Toulouse

Outreach

XMU-China

Peking University

NanJin University

France

Toulouse

USA Purdue

Outreach

AcknowledgementProf. Hasty and his student Arthur Prindle, Department of Bioengineering, University of California, San Diego, La Jolla, California.Graduates in Lab 571,Lab 580 and Lab 119, College of Chemistry and Chemical Engineering.Prof. Ningshao Xia and his graduates, School of Life Sciences, Xiamen University.Prof. Shoufa Han and his graduatesInstitute of Urban Environment, Chinese Academy of Sciences.Fujian Chemical Society and Prof. Yihui Chen. High school students from all over Fujian Province taking part in our Human Practice. Chinese University of HKPeking iGEM 2013XMU-Software

Thanks for your attention!