- Email: [email protected]
Microbial strain improvement with femtosecond laser manipulation
Abstracts / Journal of Biotechnology 136S (2008) S345–S355 Zhang, Y.X., Perry, K., Vinci, V.A., Powell, K., Stemmer, W.P., Cardayre, S.B., 2002. Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature 415, 644–646.
S349
V2-P-006 Solid cultivation of Nostoc flagelliforme cells and their growth characteristics
doi:10.1016/j.jbiotec.2008.07.798
Shiru Jia ∗ , Sijun Yue
V2-P-005
Tianjin Key Lab of Industrial Microbiology, Tianjin University of Science and Technology, Tianjin 300457, China
Microbial strain improvement with femtosecond laser manipulation
E-mail address: [email protected] (S. Jia).
Jixian Gong 1,∗ , Xueming Zhao 1 , Qirong Xing 2 1
School of Chemical Engineering and Technology, Edinburgh-Tianjin Joint Centre for Systems Biology and Synthetic Biology, Tianjin University, Tianjin 300072, China 2 School of Precision Instrument & Optoelectronics Engineering, Tianjin University, Tianjin 300072, China E-mail address: [email protected] (X. Zhao).
Single-cell techniques have been crucial in solving the problem of high-throughput screening of microbial strain. Femtosecond laser has been applied to biological research at single-cell level for its advantages of ultra-high temporal and spatial resolutions (Vogel et al., 2005; Tirlapur and König, 2002; Watanabe et al., 2004). The deficiencies in cell fusion with traditional methods can be meliorated by using femtosecond laser pulses. In this work, femtosecond laser was employed to induce cell fusion. Phaffia rhodozyma, which is one of the main sources for nature astaxanthin, was selected as target sample. The protoplasts obtained with enzyme were placed in a manipulation chamber. A pair of effectively contacted cells was selected, and then femtosecond laser beam with 1.38 × 104 W was shot straight at the interface between the two contacted cells. The irradiation of the interface by femtosecond laser lasted 0.25 s. The cell fusion became visible about 20 min later. At the end of this time interval, the two original cells merged into one cell. The similar experiments were repeated 5 times at different laser power and each time 20 pairs of new cells were used. These results strongly suggest the existence of a threshold peak power for induced cell fusion, which is estimated as approximately 1.25 × 104 W. The fusion rate at this power level was 80%. In conclusion, cell fusion induced by femtosecond laser pulses was achieved at single-cell level. Femtosecond laser-induced cell fusion was manipulated at single-cell resolution and hybrid cells were facilitated for screening. The technology of cell fusion with femtosecond laser manipulation at single-cell resolution has potential applications in microbial strain improvement. Acknowledgement Supported by NSFC-20536040, 973-2007CB707802 References Tirlapur, U.K., König, K., 2002. Targeted transfection by femtosecond laser. Nature 418, 290–291. Vogel, A., Noack, J., Hüttman, G., Paltauf, G., 2005. Mechanisms of femtosecond laser nanosurgery of cells and tissues. Appl. Phys. B 81, 1015–1047. Watanabe, W., Arakawa, N., Matsunaga, S., Matsunaga, S., Higashi, T., Fukui, K., Itoh, K., 2004. Femtosecond laser disruption of subcellular organelles in a living cell. Opt. Express 12, 4203–4213.
doi:10.1016/j.jbiotec.2008.07.799
The solid cultivation of Nostoc flagelliforme cells obtained from liquid culture was studied and the result showed that the free-living N. flagelliforme cells can grow on solid sand. Their growth rate on the sand is faster than that of the trichome (Su et al., 2005). The photosynthesis and respiration of the dehydrated cells recovered rapidly within 24 h of rehydration, which meant that N. flagelliforme cells are tolerant to drought just like the wild trichome. The temperature tolerance experiment showed that the respiratory and photosynthetic rates of N. flagelliforme cells in solid culture are higher than those in liquid culture, which indicated that the solid-culturedcells are more resistant to high temperature though the respiratory and photosynthetic rates in both cultivation styles decreased with the increase of the temperature. There was no significant difference in the alternating dry-wet condition for N. flagelliforme cells starting from the 10th, 20th and 30th days of solid cultivation respectively, which revealed that N. flagelliforme cells are able to adapt to the humidity change of the environment quickly. The result also showed that the solid cultivation of N. flagelliforme cells can improve the soil quality in water permeability, water retention and surface crusting. In the solid cultivation, N. flagelliforme cells obtained from liquid culture exhibited adaptability to draught similar to the wild N. flagelliforme trichome. Reference Su, J.Y., Jia, S.R., Qiao, C.S., Jung, G.K., 2005. Culture of Nostoc flagelliforme on solid medium. Korean J. Environ. Biol. 23, 135–140.
doi:10.1016/j.jbiotec.2008.07.800 V2-P-009 BestCodon: Novel software for codon bias analysis Jincong Yu, Lin Shui, Baishan Fang ∗ Key Laboratory of Industrial Biotechnology (Huaqiao University), Fujian Province University, Quanzhou 362021, China E-mail address: [email protected] (B. Fang). Each organism has unique codon usage. Analyzing the organism’s codon bias is helpful to comprehend the origin and evolution of codons. Especially, proper codon optimization may enhance the expression of target genes which are on heterologous expression. Before the optimization, the difference between gene donors and gene donees should be known in advance. At present, there are some softwares for the codon bias analysis based on existing sequence data, but most of them run in the Unix or Linux operating system and only have rough command line interfaces. It is inconvenient for many users. For improving that, the BestCodon have been developed, which run in the Windows platform and have a convenient graphic user interface. The software is mainly constituted of two modules. One is statistics module that codon usage tabulate (CUT) can be calculated out from fasta format files containing one or more coding sequences (CDS). The other is transformation module that CDS can be picked up and saved as fasta format files from the GenBank page files containing sequences information. Therefore,
S349
V2-P-006 Solid cultivation of Nostoc flagelliforme cells and their growth characteristics
doi:10.1016/j.jbiotec.2008.07.798
Shiru Jia ∗ , Sijun Yue
V2-P-005
Tianjin Key Lab of Industrial Microbiology, Tianjin University of Science and Technology, Tianjin 300457, China
Microbial strain improvement with femtosecond laser manipulation
E-mail address: [email protected] (S. Jia).
Jixian Gong 1,∗ , Xueming Zhao 1 , Qirong Xing 2 1
School of Chemical Engineering and Technology, Edinburgh-Tianjin Joint Centre for Systems Biology and Synthetic Biology, Tianjin University, Tianjin 300072, China 2 School of Precision Instrument & Optoelectronics Engineering, Tianjin University, Tianjin 300072, China E-mail address: [email protected] (X. Zhao).
Single-cell techniques have been crucial in solving the problem of high-throughput screening of microbial strain. Femtosecond laser has been applied to biological research at single-cell level for its advantages of ultra-high temporal and spatial resolutions (Vogel et al., 2005; Tirlapur and König, 2002; Watanabe et al., 2004). The deficiencies in cell fusion with traditional methods can be meliorated by using femtosecond laser pulses. In this work, femtosecond laser was employed to induce cell fusion. Phaffia rhodozyma, which is one of the main sources for nature astaxanthin, was selected as target sample. The protoplasts obtained with enzyme were placed in a manipulation chamber. A pair of effectively contacted cells was selected, and then femtosecond laser beam with 1.38 × 104 W was shot straight at the interface between the two contacted cells. The irradiation of the interface by femtosecond laser lasted 0.25 s. The cell fusion became visible about 20 min later. At the end of this time interval, the two original cells merged into one cell. The similar experiments were repeated 5 times at different laser power and each time 20 pairs of new cells were used. These results strongly suggest the existence of a threshold peak power for induced cell fusion, which is estimated as approximately 1.25 × 104 W. The fusion rate at this power level was 80%. In conclusion, cell fusion induced by femtosecond laser pulses was achieved at single-cell level. Femtosecond laser-induced cell fusion was manipulated at single-cell resolution and hybrid cells were facilitated for screening. The technology of cell fusion with femtosecond laser manipulation at single-cell resolution has potential applications in microbial strain improvement. Acknowledgement Supported by NSFC-20536040, 973-2007CB707802 References Tirlapur, U.K., König, K., 2002. Targeted transfection by femtosecond laser. Nature 418, 290–291. Vogel, A., Noack, J., Hüttman, G., Paltauf, G., 2005. Mechanisms of femtosecond laser nanosurgery of cells and tissues. Appl. Phys. B 81, 1015–1047. Watanabe, W., Arakawa, N., Matsunaga, S., Matsunaga, S., Higashi, T., Fukui, K., Itoh, K., 2004. Femtosecond laser disruption of subcellular organelles in a living cell. Opt. Express 12, 4203–4213.
doi:10.1016/j.jbiotec.2008.07.799
The solid cultivation of Nostoc flagelliforme cells obtained from liquid culture was studied and the result showed that the free-living N. flagelliforme cells can grow on solid sand. Their growth rate on the sand is faster than that of the trichome (Su et al., 2005). The photosynthesis and respiration of the dehydrated cells recovered rapidly within 24 h of rehydration, which meant that N. flagelliforme cells are tolerant to drought just like the wild trichome. The temperature tolerance experiment showed that the respiratory and photosynthetic rates of N. flagelliforme cells in solid culture are higher than those in liquid culture, which indicated that the solid-culturedcells are more resistant to high temperature though the respiratory and photosynthetic rates in both cultivation styles decreased with the increase of the temperature. There was no significant difference in the alternating dry-wet condition for N. flagelliforme cells starting from the 10th, 20th and 30th days of solid cultivation respectively, which revealed that N. flagelliforme cells are able to adapt to the humidity change of the environment quickly. The result also showed that the solid cultivation of N. flagelliforme cells can improve the soil quality in water permeability, water retention and surface crusting. In the solid cultivation, N. flagelliforme cells obtained from liquid culture exhibited adaptability to draught similar to the wild N. flagelliforme trichome. Reference Su, J.Y., Jia, S.R., Qiao, C.S., Jung, G.K., 2005. Culture of Nostoc flagelliforme on solid medium. Korean J. Environ. Biol. 23, 135–140.
doi:10.1016/j.jbiotec.2008.07.800 V2-P-009 BestCodon: Novel software for codon bias analysis Jincong Yu, Lin Shui, Baishan Fang ∗ Key Laboratory of Industrial Biotechnology (Huaqiao University), Fujian Province University, Quanzhou 362021, China E-mail address: [email protected] (B. Fang). Each organism has unique codon usage. Analyzing the organism’s codon bias is helpful to comprehend the origin and evolution of codons. Especially, proper codon optimization may enhance the expression of target genes which are on heterologous expression. Before the optimization, the difference between gene donors and gene donees should be known in advance. At present, there are some softwares for the codon bias analysis based on existing sequence data, but most of them run in the Unix or Linux operating system and only have rough command line interfaces. It is inconvenient for many users. For improving that, the BestCodon have been developed, which run in the Windows platform and have a convenient graphic user interface. The software is mainly constituted of two modules. One is statistics module that codon usage tabulate (CUT) can be calculated out from fasta format files containing one or more coding sequences (CDS). The other is transformation module that CDS can be picked up and saved as fasta format files from the GenBank page files containing sequences information. Therefore,