(620w) Use of Lectin-Conjugated Magnetite Nanoparticles and Magnetic Force to Screen Mutant Cyanobacteria in a Single Cell Culture System

We developed a single cell culture system to screen mutant cyanobacteria by using magnetic nanoparticles (MNPs) and magnetic force. This system could perform a single cell isolation culture with high viability compared to conventional methods.

  Cyanobacteria such as Synechococcus elongatus PCC7942 have the potential to produce biofuels directly from CO₂ [1]. To raise the productivity of biofuel, it is necessary to select the useful mutants such as strains which have high growth rate and are resistant to specific metabolic products. The general approach for screening of mutants involves single colony isolation in solid culture. However, it is inefficient for cyanobacteria with long doubling time and low colony-forming ratio due to the cell death by dehydration. Although we have previously developed the droplet culture system for single cell culture of cyanobacteria to solve the dehydration problem [2], the system was not suitable for a long term culture and a screening throughput of the system was low. Thus, more efficient single cell culture system for screening mutant cyanobacteria is still required.

  In the present study, we applied the following cell patterning method established originally by us to a single cell culture of cyanobacteria in liquid. We have developed a magnetic force-based cell patterning method for mammalian cells and succeeded in analyzing cells at a single-cell level [3-5]. In this method, we fabricated magnetite cationic liposome (MCL) in which MNPs were coated by cationic liposome and MCLs were nonspecifically incorporated by mammalian cells. Then, the magnetically labeled cells can be arrayed by using a magnetic pin-holder device which has 6400 iron pillars (each 100 µm in width and 100 µm in length) with 150 µm intervals. In order to develop MCLs to label specifically PCC7942 cells, we focused on the lectin which can recognize specific sugar chain in the outer membrane of cells because the lipopolysaccharide exists on the outer membrane of cyanobacteria. We found that LCA (Lens culinaris Agglutinin) which recognize mannose as a sugar could bind to the surface of PCC7942 cells, and then developed the LCA-conjugated MCLs. The MCL labeled PCC7942 cells were magnetically patterned at a single cell level by iron pillars of the pin-holder device, and 1600 of single cell are enable to be arrayed in a square centimeter. As a result of culturing the patterned cells for 7 days, we observed colony formation from single cell in liquid medium and not observed any harmful effect of the LCA-conjugated MCLs to the cell growth. We achieved higher colony-forming ratio by the array culture method (78.4%) than that by the conventional solid culture method (4.8%). Furthermore, strains with different properties could be distinguished in the single culture system depending on their growth in the cell array. Finally, we could selectively pick up the target cells by a glass capillary operated by a micromanipulator and subsequently perform efficient isolation culture.

  Thus, the developed system could overcome the problems of the conventional methods, such as dehydration of the cells, limitation of initial cell density, difficulty of long-term culture maintenance, and low throughput performance. It would be useful for screening mutant cyanobacteria.

References

[1] T. Kusakabe, T. Tatsuke, K. Tsuruno, Y. Hirokawa, S. Atsumi, J. C. Liao and T. Hanai, Metab. Eng., 20 (2013), 101-108.

[2] S. Arai, M. Okochi, T. Hanai and H. Honda, Biotech. Rep., 4 (2014), 151-155.

[3] K. Ino, M. Okochi, N. Konishi, M. Nakatochi, R. Imai, M. Shikida, A. Ito and H. Honda, Lab Chip, 8 (2008), 134-142

[4] M. Okochi, S. Takano, Y. Isaji, T. Senga, M. Hamaguchi, H. Honda, Lab Chip, 9 (2009), 3378-3384

[5] S. Yamamoto, M.M. Hotta, M. Okochi, H. Honda, PLoS ONE, 9 (2014), e103502