TMU-Tokyo, from Tokyo Metropolitan University

～Extend utilization of BioBricks～

October 5th, 2013

iGEM Asia Jamboree

Background

transformation

In the past

In our project

pSB1C3

pSB1C3

BioBrick

DNA

BioBrick

PCR products

Using Homologous Recombination System!

Background

1. You don’t have to care the incompatibility of plasmids.

2. You can insert long fragment over 15kb in E. coli genome.

“Breakthrough” is better than the conventional method.

3. Once you inserted the fragment in the genome, this fragment is hardly lost.

1k bp 1k bp

Background -red recombination system-

λ red homologous recombination

E . coli homologous recombination

40 bp 40 bp

homology homology

Brief Summary

In this year, we tried to・・・

→Insert DNA fragments such as BioBricks into E. coli genome. →Create new method of genome engineering to expand the utilization of BioBricks.

And we really

→created new method, “Breakthrough”. →inserted 4 fragments in E. coli genome.

Outline

“Breakthrough” (1) Institution Materials (2) First Strategy

(2)Results (1) Over view

Application example

(3) Second strategy

“Breakthrough” -Overview-

(2) First Strategy：

(3) Second strategy：For DNA fragments

(1) Institution material for others

For BioBrick in pSB1C3

・Modified plasmid vector

・List of primer sets

・Experimental protocol

(1) Institution Manual

• Make the new method to insert in genome of E. coli

• Make the Primer sequence list for red recombination http://2013.igem.org/File:TMUNew_standard_2.xls

(2) First strategy

pSB1C3

CamR BioBricks

0.2kb

CamR BioBricks

PCR

Genome

E.coli K12 strain MG1655 red

Transformation Homologous recombination

CmR BioBricks

(2) First strategy

Selection marker New BioBricks

New BioBricks Selection marker

Overlap extention PCR

(2) second strategy

New BioBricks

Selection marker

New BioBricks

Selection marker

Addition of Homologous sequence by PCR

(2) second strategy

New BioBricks

Selection marker

RED homologous recombination

transformation

Cloning into pSB1C3

(2) second strategy

pSB1C3

EcoRⅠ

prefix XbaⅠ PstⅠ SpeⅠ

suffix

Our new part: Modified plasmid vector

XbaⅠ SpeⅠ BB_K1015013

SmaⅠ PvuⅡ NruⅠ NruⅠ PvuⅡ SmaⅠ

Km

BB_K1015013

SmaⅠ PvuⅡ NruⅠ NruⅠ PvuⅡ SmaⅠ

Km

Our new part: Modified plasmid vector

Breakthrough -Overview-

(2) First Strategy：

(3) Second strategy：For DNA fragments

(1) Institution material for others

For BioBrick in pSB1C3

・Modified plasmid vector

・List of primer sets ・Experimental protocol

Overview -Integration targets-

(BBa_K1015014) (BBa_K1015015)

(BBa＿K1015016)

(BBa_K1015017)

Flow chart of the experiments

<Fragment 1> <Fragment 2> <Fragment3>

1655 RED+Fragment2 1655 RED+Fragment3-1 1655 RED+Fragment1

P1 +fragment1

1655 RED +

Fragment3-1&3-2

Fragment1 Fragment2 Fragment3-1

Fragment3-2

MG1655 Pythagorean Devices

P1 +fragment2

P1 +fragment3

transformation P1 transduction P1 preparation

FRT lacI FRT lacZ

IRR IRL flp

ptet

How does it work?

pBAD hbiF

arabinose

plac FRT

β- galactosidase

Results -overview-

(2) X-gal assey

(3)-2 fragment assey -deletion-

(1) Genome insertion assey

(3)-1 fragment assey -inversion-

↑ Control (1655 red)

Primer Primer

(1) genome insertion assay

BB_K1015014

(1) genome insertion assay

↑ Control (1655 red)

Primer Primer

↑ Control (1655 red fragment3 )

BB_K1015015

BB_K1015016

Primer Primer

(2) X-gal assey

Control (MG1655)

Experimental group

(3)-1 Check of inversion between IRR and IRL

<Pythagorean devises>

IRR IRL flp

ptet Primer Primer

PCR Products

IRR IRL flp

ptet Primer Primer

PCR Products

⇒Fragment 1&2 are working

<only fragment 1 >

(3)-2 Check of deletion between FRTs

FRT lacI FRT lacZ plac FRT

FRT lacI FRT lacZ plac

Primer Primer

Primer Primer

(3)-2 over expression of FLP

FRT lacI FRT lacZ plac FRT

β- galactosidase

FLP FLP FLP

pFTGT

(3)-2 over expression of FLP

Control (MG1655)

Experimental group

Conclusions

New protocols to expand utilization of BioBricks were established for E. coli genome engineering.

Modified useful pSB1C3 was developed (BB_K1015013).

Four DNA fragments were successfully inserted in a genome of E. coli.

Our “Pythagorean” device really worked.

Human practice

・iGEM Japan Meet Up 2013

・Contribution to TUPLS-Tokyo

・Interchange with Macquarie_University team

7 teams participated

・Science AGORA 2012 in Tokyo

・Lecture of the Synthetic Biology. at Tamagawagakuen high school at Ohyugakuen high school

(Total 238 students listened. )

Promote Synthetic Biology

Collaboration

Checklist for Safely experiment

experoment

Pre-experiment

During-experiment

Post-experiment

Achievement

Established new and convenient method to extend

utilize of Biobricks. Created a new and convenient part (BBa_K1015013)

for this method. This part extends utilize of pSB1C3. Shared above method for all other iGEMers. With our method, we succeeded to insert all of our

parts in E. coli genome. Our “Pythagorean” device really worked!!

Support from the following laboratories

Tokyo Metoropolitan University Division of Biological Sciences

Acknowledgement

Environmental Microbiology Plant Hormone Mechanism Development Programs Cellular Biochemistry Molecular Genetics

Sponser

Acknowledgement

Support from the following laboratories Tokyo Metoropolitan University Division of Biological Sciences

Environmental Microbiology Plant Hormone Mechanism Development Programs Cellular Biochemistry Molecular Genetics

RED recombination System

gam exo beta

gam beta exo

recBCD

Breakthrough (4) P1 transduction Genome assembly

Genome of E . coli

1655 red

BioBrick1

P1 bacteriophage infection

Breakthrough (4) P1 transduction Genome assembly

Breakthrough (4) P1 transduction Genome assembly

BioBrick2

P1 bacteriophage transduction

Breakthrough (4) P1 transduction Genome assembly

BioBrick2 BioBrick1

P1 bacteriophage transduction

Results fragment assay

Fragment 3-1 3-2

Fragment 1 Fragment 2

Results inversion/deletion assay

Fragment3 +pFTGT

Fragment3

Fragment 1+2+3 1~3 LB+ara 4~6 LB 7 Fragment 1 (control)

1 2 3 4 5 6 7

Results genome insertion assay

BB_K1015014

Primer Primer

Hit

↑ Control (1655 red)

Results genome insertion assay

Primer Primer

pBAD

BB_K1015015

↑ Control (1655 red)

Primer Primer

↑ Control (1655 red fragment3 )

X-GAL assay

Fragnent 3-1+3-2 + pFTGT

Fragnent 3-1+3-2

Results fragment2 BB_K1015015

←PCR Product

Results fragment3-Ap BB_K1015016

←PCR Product

Results fragment3-Cm BB_K1015017

PCR Product→

Implementation

6. Repression of plac becomes off by deleting of lacI gene and then β-gal comes to be expressed.

plac lacZ lacY lacA

Β-gal

Horizontal transmission

Plasmid

It is highly possible that horizontal transmission occurs.

Implication

Background - overlap extention PCR-

1st PCR

Primer to amplify B added 40mer Overlap sequence of A

Primer to amplify A added 40mer Overlap sequence of B

A

B

2nd PCR

Background - overlap extention PCR-

Devices fragment 3

FRT Amp lacI FRT Cm

1. A large quantity of lacI is developed by the lacI gene.

Human & Safety

Collaboration

iGEM Japan Meet Up 2013

・TMU-Tokyo (Host) ・UT-Tokyo ・Kyoto ・Tokyo_Tech 2013 ・Tokyo-NoKogen ・Hokkaido_U ・TUPLS-Tokyo

Contribution to TUPLS-Tokyo

Human & Safety

Promote Synthetic Biology

Science AGORA 2012 in Tokyo

Lecture about the Synthetic Biology