Limits...
Optimization of bacterial plasmid transformation using nanomaterials based on the Yoshida effect.

Tan H, Fu L, Seno M - Int J Mol Sci (2010)

Bottom Line: Only a few transformants could be obtained even when 100 ng of plasmid pET15b was used, and a successful result seemed difficult to repeat.Meanwhile, the results could also be reproduced well.Meanwhile, compared with the mechanism previously reported, we verified quite a different principle for the mechanism responsible for such a transformation.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Department, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; E-Mail: hdt@dicp.ac.cn.

ABSTRACT
With the help of sepiolite, a unique method for transforming DNA into bacteria, based on the Yoshida effect, has been developed recently. However, we confronted many problems when this newest method was tried. Only a few transformants could be obtained even when 100 ng of plasmid pET15b was used, and a successful result seemed difficult to repeat. To address this problem, we optimized the operating method and could achieve about 15,000 transformants using the same amount of plasmid, which could match the efficiency gained using the calcium chloride transformation method. Meanwhile, the results could also be reproduced well. In the same way, carbon nanotubes were used to attain more than 15,000 transformants in the same situation. Therefore, the transformation method could be extended to other nanomaterials. Meanwhile, compared with the mechanism previously reported, we verified quite a different principle for the mechanism responsible for such a transformation. In sum, this unique transformation can be developed to become the third widely-used transformation method in laboratories in addition to the chemical method and electroporation.

Show MeSH

Related in: MedlinePlus

After absorbing GFPuv, the shape of sepiolite is observed by fluorescence microscope. There are three kinds of sepiolite: untreated sepiolite (sepiolite 1), the sepiolite collected from the supernatants (sepiolite 2) and ultrasonicated sepiolite (sepiolite 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC3100829&req=5

f4-ijms-11-04962: After absorbing GFPuv, the shape of sepiolite is observed by fluorescence microscope. There are three kinds of sepiolite: untreated sepiolite (sepiolite 1), the sepiolite collected from the supernatants (sepiolite 2) and ultrasonicated sepiolite (sepiolite 3).

Mentions: Additionally, we selected two other sepiolites (sepiolite 2 and 3) with more fibers compared with sepiolite 1 (the untreated sepiolite), see Figure 4. Sepiolite 2 is the sepiolite collected from the supernatants and sepiolite 3 is ultrasonicated sepiolite (Figure 4). According to the mechanism supplied by previous reports [4], sepiolite with more nanofibers will produce higher-efficiency transformation. Conversely, the transformation efficiency was reduced greatly, and even failed to produce transformants when ultrasonicated sepiolite was used, while the transformation efficiency was the highest when the sepiolite was untreated (Figure 2, samples 1–3). In the same way, using CNTs produced similar results. These results suggested that the fibers alone hardly influenced the success of the plasmid transformation. Heterogeneous DNA still could enter the cell even without absorbing on the sepiolite fibers. The transformation might need the larger size of sepiolite to destroy the cell wall for the plasmids to enter the cell envelope through the temporary nanochannels formed in the cell membrane.


Optimization of bacterial plasmid transformation using nanomaterials based on the Yoshida effect.

Tan H, Fu L, Seno M - Int J Mol Sci (2010)

After absorbing GFPuv, the shape of sepiolite is observed by fluorescence microscope. There are three kinds of sepiolite: untreated sepiolite (sepiolite 1), the sepiolite collected from the supernatants (sepiolite 2) and ultrasonicated sepiolite (sepiolite 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3100829&req=5

f4-ijms-11-04962: After absorbing GFPuv, the shape of sepiolite is observed by fluorescence microscope. There are three kinds of sepiolite: untreated sepiolite (sepiolite 1), the sepiolite collected from the supernatants (sepiolite 2) and ultrasonicated sepiolite (sepiolite 3).
Mentions: Additionally, we selected two other sepiolites (sepiolite 2 and 3) with more fibers compared with sepiolite 1 (the untreated sepiolite), see Figure 4. Sepiolite 2 is the sepiolite collected from the supernatants and sepiolite 3 is ultrasonicated sepiolite (Figure 4). According to the mechanism supplied by previous reports [4], sepiolite with more nanofibers will produce higher-efficiency transformation. Conversely, the transformation efficiency was reduced greatly, and even failed to produce transformants when ultrasonicated sepiolite was used, while the transformation efficiency was the highest when the sepiolite was untreated (Figure 2, samples 1–3). In the same way, using CNTs produced similar results. These results suggested that the fibers alone hardly influenced the success of the plasmid transformation. Heterogeneous DNA still could enter the cell even without absorbing on the sepiolite fibers. The transformation might need the larger size of sepiolite to destroy the cell wall for the plasmids to enter the cell envelope through the temporary nanochannels formed in the cell membrane.

Bottom Line: Only a few transformants could be obtained even when 100 ng of plasmid pET15b was used, and a successful result seemed difficult to repeat.Meanwhile, the results could also be reproduced well.Meanwhile, compared with the mechanism previously reported, we verified quite a different principle for the mechanism responsible for such a transformation.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Department, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; E-Mail: hdt@dicp.ac.cn.

ABSTRACT
With the help of sepiolite, a unique method for transforming DNA into bacteria, based on the Yoshida effect, has been developed recently. However, we confronted many problems when this newest method was tried. Only a few transformants could be obtained even when 100 ng of plasmid pET15b was used, and a successful result seemed difficult to repeat. To address this problem, we optimized the operating method and could achieve about 15,000 transformants using the same amount of plasmid, which could match the efficiency gained using the calcium chloride transformation method. Meanwhile, the results could also be reproduced well. In the same way, carbon nanotubes were used to attain more than 15,000 transformants in the same situation. Therefore, the transformation method could be extended to other nanomaterials. Meanwhile, compared with the mechanism previously reported, we verified quite a different principle for the mechanism responsible for such a transformation. In sum, this unique transformation can be developed to become the third widely-used transformation method in laboratories in addition to the chemical method and electroporation.

Show MeSH
Related in: MedlinePlus