Rapid in vitro method to assemble and transfer DNA fragments into the JCVI-syn3B minimal synthetic bacterial genome through Cre/loxP system
JCVI-syn3B (syn3B), a minimal synthetic bacterium that only possesses essential genes, facilitates the examination of heterogeneous gene functions in minimal life. Conventionally, Escherichia coli is used to construct DNA fragments for gene transfer into the syn3B genome through Cre/loxP system. How...
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| Format: | Article |
| Language: | English |
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The Biophysical Society of Japan
2024-12-01
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| Series: | Biophysics and Physicobiology |
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| Online Access: | https://doi.org/10.2142/biophysico.bppb-v21.0024 |
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| author | Atsuko Uenoyama Hana Kiyama Mone Mimura Makoto Miyata |
| author_facet | Atsuko Uenoyama Hana Kiyama Mone Mimura Makoto Miyata |
| author_sort | Atsuko Uenoyama |
| collection | DOAJ |
| description | JCVI-syn3B (syn3B), a minimal synthetic bacterium that only possesses essential genes, facilitates the examination of heterogeneous gene functions in minimal life. Conventionally, Escherichia coli is used to construct DNA fragments for gene transfer into the syn3B genome through Cre/loxP system. However, the construction process is challenging and time-consuming due to various issues, including the inhibition of E. coli growth and unexpected recombination, especially with AT-rich DNA sequences such as those found in Mycoplasma genes. Therefore, in this study, we aimed to develop a new transformation method to overcome these issues. We assembled the vector and target DNA fragments using an in vitro homologous recombination system and subsequently transferred the products into the syn3B genome. We obtained approximately 103~104 recombinant colonies per milliliter of the original culture in eight days, which is four days shorter than the conventional period, without any recombination issues, even for AT-rich DNA. This method may be applicable to other gene manipulation systems based on Cre/loxP system. |
| format | Article |
| id | doaj-art-a6d0c5ee74094393b6a83d421f5bd802 |
| institution | Kabale University |
| issn | 2189-4779 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | The Biophysical Society of Japan |
| record_format | Article |
| series | Biophysics and Physicobiology |
| spelling | doaj-art-a6d0c5ee74094393b6a83d421f5bd8022025-01-09T10:11:42ZengThe Biophysical Society of JapanBiophysics and Physicobiology2189-47792024-12-012110.2142/biophysico.bppb-v21.0024Rapid in vitro method to assemble and transfer DNA fragments into the JCVI-syn3B minimal synthetic bacterial genome through Cre/loxP systemAtsuko Uenoyama0Hana Kiyama1Mone Mimura2Makoto Miyata3Graduate School of Science, Osaka Metropolitan University, Osaka 558-8585, JapanGraduate School of Science, Osaka Metropolitan University, Osaka 558-8585, JapanGraduate School of Science, Osaka Metropolitan University, Osaka 558-8585, JapanGraduate School of Science, Osaka Metropolitan University, Osaka 558-8585, JapanJCVI-syn3B (syn3B), a minimal synthetic bacterium that only possesses essential genes, facilitates the examination of heterogeneous gene functions in minimal life. Conventionally, Escherichia coli is used to construct DNA fragments for gene transfer into the syn3B genome through Cre/loxP system. However, the construction process is challenging and time-consuming due to various issues, including the inhibition of E. coli growth and unexpected recombination, especially with AT-rich DNA sequences such as those found in Mycoplasma genes. Therefore, in this study, we aimed to develop a new transformation method to overcome these issues. We assembled the vector and target DNA fragments using an in vitro homologous recombination system and subsequently transferred the products into the syn3B genome. We obtained approximately 103~104 recombinant colonies per milliliter of the original culture in eight days, which is four days shorter than the conventional period, without any recombination issues, even for AT-rich DNA. This method may be applicable to other gene manipulation systems based on Cre/loxP system.https://doi.org/10.2142/biophysico.bppb-v21.0024homologous recombinationtransformation |
| spellingShingle | Atsuko Uenoyama Hana Kiyama Mone Mimura Makoto Miyata Rapid in vitro method to assemble and transfer DNA fragments into the JCVI-syn3B minimal synthetic bacterial genome through Cre/loxP system Biophysics and Physicobiology homologous recombination transformation |
| title | Rapid in vitro method to assemble and transfer DNA fragments into the JCVI-syn3B minimal synthetic bacterial genome through Cre/loxP system |
| title_full | Rapid in vitro method to assemble and transfer DNA fragments into the JCVI-syn3B minimal synthetic bacterial genome through Cre/loxP system |
| title_fullStr | Rapid in vitro method to assemble and transfer DNA fragments into the JCVI-syn3B minimal synthetic bacterial genome through Cre/loxP system |
| title_full_unstemmed | Rapid in vitro method to assemble and transfer DNA fragments into the JCVI-syn3B minimal synthetic bacterial genome through Cre/loxP system |
| title_short | Rapid in vitro method to assemble and transfer DNA fragments into the JCVI-syn3B minimal synthetic bacterial genome through Cre/loxP system |
| title_sort | rapid in vitro method to assemble and transfer dna fragments into the jcvi syn3b minimal synthetic bacterial genome through cre loxp system |
| topic | homologous recombination transformation |
| url | https://doi.org/10.2142/biophysico.bppb-v21.0024 |
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