Download - iGEM Asia Jamboree
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