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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.

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The optimization of DNA transformation for Escherichia coli DH5α based on sepiolite. (1–2) The effect of sepiolite buffer on transformation frequency: Sepiolite buffer was used in sample 1 and LB medium was used directly in sample 2; (3–5) The effect of varying cell concentration: OD600 nm = 200, 20 and 2 in sample 3, 4 and 5 respectively; (6–8) The effect of percent content of sepiolite: 1%, 0.1% and 0.01% in sample 6, 7 and 8 respectively; (9–12) The effect of time of streaking to plate the transformed cells: 10, 20, 40 and 100 s in sample 9, 10, 11 and 12 respectively; (13–15) The effect of washing treatment with ddH2O on transformation frequency. Sample 13, DNA-binding sepiolite. The DNA-binding sepiolite was washed once and twice by ddH2O in sample 14 and 15; (16–18) The effect of RNA competition on transformation frequency. Sample 16, no RNA was added. For sample 17 and 18, 300 ng and 3 μg small RNA was added in 50 μL transforming solution.
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f1-ijms-11-04962: The optimization of DNA transformation for Escherichia coli DH5α based on sepiolite. (1–2) The effect of sepiolite buffer on transformation frequency: Sepiolite buffer was used in sample 1 and LB medium was used directly in sample 2; (3–5) The effect of varying cell concentration: OD600 nm = 200, 20 and 2 in sample 3, 4 and 5 respectively; (6–8) The effect of percent content of sepiolite: 1%, 0.1% and 0.01% in sample 6, 7 and 8 respectively; (9–12) The effect of time of streaking to plate the transformed cells: 10, 20, 40 and 100 s in sample 9, 10, 11 and 12 respectively; (13–15) The effect of washing treatment with ddH2O on transformation frequency. Sample 13, DNA-binding sepiolite. The DNA-binding sepiolite was washed once and twice by ddH2O in sample 14 and 15; (16–18) The effect of RNA competition on transformation frequency. Sample 16, no RNA was added. For sample 17 and 18, 300 ng and 3 μg small RNA was added in 50 μL transforming solution.

Mentions: We confronted many problems when we repeated the procedure provided in a previous report [4], and only a few transformants could be obtained although 100 ng of plasmid pET15b was used (Figure 1, sample 6). Moreover, the work was challenging to repeat. Therefore, we optimized various parameters for such a transformation method. We found that the sepiolite buffer was not necessary since the result was no better that when LB medium was used (Figure 1, samples 1 and 2). Furthermore, the cultured cells could be transformed by plasmids in LB medium directly. On the other hand, 0.01% sepiolite was too low to be successful for such a transformation (Figure 1, samples 6–8). Additionally, the time of streaking the plate to spread the transformed cells could affect the efficiency of plasmid transformation greatly (Figure 1, samples 9–12).


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

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

The optimization of DNA transformation for Escherichia coli DH5α based on sepiolite. (1–2) The effect of sepiolite buffer on transformation frequency: Sepiolite buffer was used in sample 1 and LB medium was used directly in sample 2; (3–5) The effect of varying cell concentration: OD600 nm = 200, 20 and 2 in sample 3, 4 and 5 respectively; (6–8) The effect of percent content of sepiolite: 1%, 0.1% and 0.01% in sample 6, 7 and 8 respectively; (9–12) The effect of time of streaking to plate the transformed cells: 10, 20, 40 and 100 s in sample 9, 10, 11 and 12 respectively; (13–15) The effect of washing treatment with ddH2O on transformation frequency. Sample 13, DNA-binding sepiolite. The DNA-binding sepiolite was washed once and twice by ddH2O in sample 14 and 15; (16–18) The effect of RNA competition on transformation frequency. Sample 16, no RNA was added. For sample 17 and 18, 300 ng and 3 μg small RNA was added in 50 μL transforming solution.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-ijms-11-04962: The optimization of DNA transformation for Escherichia coli DH5α based on sepiolite. (1–2) The effect of sepiolite buffer on transformation frequency: Sepiolite buffer was used in sample 1 and LB medium was used directly in sample 2; (3–5) The effect of varying cell concentration: OD600 nm = 200, 20 and 2 in sample 3, 4 and 5 respectively; (6–8) The effect of percent content of sepiolite: 1%, 0.1% and 0.01% in sample 6, 7 and 8 respectively; (9–12) The effect of time of streaking to plate the transformed cells: 10, 20, 40 and 100 s in sample 9, 10, 11 and 12 respectively; (13–15) The effect of washing treatment with ddH2O on transformation frequency. Sample 13, DNA-binding sepiolite. The DNA-binding sepiolite was washed once and twice by ddH2O in sample 14 and 15; (16–18) The effect of RNA competition on transformation frequency. Sample 16, no RNA was added. For sample 17 and 18, 300 ng and 3 μg small RNA was added in 50 μL transforming solution.
Mentions: We confronted many problems when we repeated the procedure provided in a previous report [4], and only a few transformants could be obtained although 100 ng of plasmid pET15b was used (Figure 1, sample 6). Moreover, the work was challenging to repeat. Therefore, we optimized various parameters for such a transformation method. We found that the sepiolite buffer was not necessary since the result was no better that when LB medium was used (Figure 1, samples 1 and 2). Furthermore, the cultured cells could be transformed by plasmids in LB medium directly. On the other hand, 0.01% sepiolite was too low to be successful for such a transformation (Figure 1, samples 6–8). Additionally, the time of streaking the plate to spread the transformed cells could affect the efficiency of plasmid transformation greatly (Figure 1, samples 9–12).

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