Introduction

I'd like to thank the organizers for this opportunity. In the past, I was involved in ImPACT, impulsing paradigm change through disruptive technologies program. The aim of our program -- bio-innovation through artificial cell reactor. Using a microreactor array, here. The project can be broken into 3 subprojects. We aim to enhance the ... of a single-molecule reaction.

Create to synthesize the function of protein, and ... finally, we aim to proliferate the.. such as is a ... and through.. and inside of our reactor. Today my attention will be on the proliferate project.

Masayuki Su'etsugu, Rikkyo University and myself. Genome replication and gene annealing by a cell-free in vitro cloning method. And my project focus was on Creation of artificial cells by fusing bacteria and microdevices-- technologies for genome exchange.

Cell-free cloning of large circular DNA

Current popular techniues about 3 days. Furthermore, we developed a ... gene fragment assembly, we lead to, ... this approach reduces exposure. We also, because we demonstrate it with a general application, we prove that it can handle large DNA. In his slide, I am showing in vitro reconstitution of replication "cycle" of ecoli chromosome. Replication cycle reacion - RCR. Auonomous repetition of the oriC-replication cycle at 30 degrees celsius.

DNA amplificiation in RCR

...

Amplification of several hundred kb DNA by RCR as covalently closed circles

It's possible to do this up to 200 kb in size. We have achieved 350 kb. We are now working on megabase size DNA.

Gene assembly method

Two-step reaction to produce circular DNA from multiple fragments, which are ligated via overlap ends, and then RCR. The assembly step is a 30 minute isothermal reaction. Specific amplification of circular assembly. Even if the yield of the ligation reaction is low, and cloning can be quite high

Cell-free in vitro cloning kit

Scheduled for free distribution just ask: kazuhito@nojilab.t.u-tokyo.ac.jp

Creation of artificial cells by fusing bacteria and microdevices

Introductio nof synthetic mateiral into bacteria, fuse the device and bacteria to prepare an artificial cell.

Membrane fusion technique.

Arrayed lipid bilayer chamber (ALBiC)

Bilayer production efficiency is over 99%. Encapsulate dye in bilayer chamber. We reconsittute alpha-hemolsyin in bilayer chamber. Passive transport was observed. Next, we confirm that the bilayer membrane had .. function. We observe.. alpha-hemolysin in bilayer chamber. We can verify it by difufsion of fluorescent dye. It decreases over time. We confirmed that we did not impair it.

Fusion of EP and ALBiC (hybrid cell)

I'd like to show you a vide oof the fusion. In some hybrid cells, the addition of ATP increased the synthesis of GFP.

This graph shows the fluorescence intensity of the hybrid cells. The x-axis is a presents fluorecence from the beginning of the experiment, y-axis is 3 hour interval.

Beta-gal expression in DNA plasmid

We confirmed beta-gal expression in plasmid. SPiDER.

Does regeneration occur in hybrid cells?

You can see the fusion. And here you see the hybrid cell engage with the reactor like this. In this red circle, you can see the glow spike object like this, and here. Finally, in this movie, you can see the small part in the hybrid cell. This observation demonstrates regeneration in the microdevice.

Conclusion

New system for replication and propagation of the ecoli genome.

Cell-free cloning gene annealing in the absence of ecoli

Successful integration of ecoli and microdevice

Bacteria regeneration in hybrid cell (although only slightly)

Q&A

Q: In vitro cloning kit, can it replace in vivo cloning? Have you shown you can go to a limiting dilution of single plasmids? With in vivo cloning you get one plasmid per cell.

A: Contamination is a problem, yes. Difficult to remove the contamination.

Q: ... what's the difference between volume in incoming ecoli cells, and the bioreactor cells? It's surprising you get that much expression, it's going to limit your ability to produce hybrid cells.

A: The bioreactor is tiny. It's roughly the same volume. Ecoli size is about 3 or 4 micrometer diameter. My device ... just..

Thank you.