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Registro Completo |
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Biblioteca(s): |
Embrapa Recursos Genéticos e Biotecnologia. |
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Data corrente: |
26/05/2022 |
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Data da última atualização: |
18/01/2023 |
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Tipo da produção científica: |
Artigo em Periódico Indexado |
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Autoria: |
SAKAI, A.; DEICH, C. R.; NELISSEN, F. H. T.; JONKER, A. J.; BITTENCOURT, D. M. de C.; KEMPES, C. P.; WISE, K. S.; HEUS, H. A.; HUCK, W. T. S.; ADAMALA, K. P.; GLASS, J. I. |
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Afiliação: |
ANDREI SAKAI, Institute for Molecules and Materials, Radboud University, Netherlands.; CHRISTOPHER R. DEICH, University of Minnesota, Minneapolis, USA.; FRANK H. T. NELISSEN, Institute for Molecules and Materials, Radboud University, Netherlands.; AAFKE J. JONKER, Institute for Molecules and Materials, Radboud University, Netherlands.; DANIELA MATIAS DE C BITTENCOURT, Cenargen; CHRISTOPHER P. KEMPES, Santa Fe Institute, Santa Fe, USA.; KIM S. WISE, The J. Craig Venter Institute, La Jolla, USA.; HANS A. HEUS, Institute for Molecules and Materials, Radboud University, Netherlands.; WILHELM T. S. HUCK, Institute for Molecules and Materials, Radboud University, Netherlands.; KATARZYNA P. ADAMALA, University of Minnesota, Minneapolis, USA.; JOHN I. GLASS, The J. Craig Venter Institute, La Jolla, USA. |
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Título: |
Traditional protocols and optimization methods lead to absent expression in a mycoplasma cell-free gene expression platform. |
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Ano de publicação: |
2022 |
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Fonte/Imprenta: |
Synthetic Biology, v. 7, n. 1, p. 1-14, 2022. |
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DOI: |
https://doi.org/10.1093/synbio/ysac008 |
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Idioma: |
Inglês |
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Conteúdo: |
Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a Mycoplasma bacterium-based CFE system using lysates of the genomeminimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative Mycoplasma capricolum (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of E. coli. Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of in vitro transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNAse activity, yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned E. coli cell free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNAse activity. However, it is not clear which gene encodes the ribonuclease. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field. MenosCell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a Mycoplasma bacterium-based CFE system using lysates of the genomeminimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative Mycoplasma capricolum (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of E. coli. Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of in vitro transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNAse activity, yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned E. coli cell free extracts by degrading ribosomal RNAs, indica... Mostrar Tudo |
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Palavras-Chave: |
Cell-free expression system; In vitro transcription; In vitro translation. |
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Thesaurus Nal: |
Mycoplasma; Ribonucleases. |
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Categoria do assunto: |
-- |
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URL: |
https://ainfo.cnptia.embrapa.br/digital/bitstream/item/240094/1/ysac008.pdf
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Marc: |
LEADER 02749naa a2200313 a 4500
001 2143465
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024 7 $ahttps://doi.org/10.1093/synbio/ysac008$2DOI
100 1 $aSAKAI, A.
245 $aTraditional protocols and optimization methods lead to absent expression in a mycoplasma cell-free gene expression platform.$h[electronic resource]
260 $c2022
520 $aCell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a Mycoplasma bacterium-based CFE system using lysates of the genomeminimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative Mycoplasma capricolum (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of E. coli. Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of in vitro transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNAse activity, yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned E. coli cell free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNAse activity. However, it is not clear which gene encodes the ribonuclease. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field.
650 $aMycoplasma
650 $aRibonucleases
653 $aCell-free expression system
653 $aIn vitro transcription
653 $aIn vitro translation
700 1 $aDEICH, C. R.
700 1 $aNELISSEN, F. H. T.
700 1 $aJONKER, A. J.
700 1 $aBITTENCOURT, D. M. de C.
700 1 $aKEMPES, C. P.
700 1 $aWISE, K. S.
700 1 $aHEUS, H. A.
700 1 $aHUCK, W. T. S.
700 1 $aADAMALA, K. P.
700 1 $aGLASS, J. I.
773 $tSynthetic Biology$gv. 7, n. 1, p. 1-14, 2022.
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Embrapa Recursos Genéticos e Biotecnologia (CENARGEN) |
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| 1. |  | SAKAI, A.; DEICH, C. R.; NELISSEN, F. H. T.; JONKER, A. J.; BITTENCOURT, D. M. de C.; KEMPES, C. P.; WISE, K. S.; HEUS, H. A.; HUCK, W. T. S.; ADAMALA, K. P.; GLASS, J. I. Traditional protocols and optimization methods lead to absent expression in a mycoplasma cell-free gene expression platform. Synthetic Biology, v. 7, n. 1, p. 1-14, 2022.| Tipo: Artigo em Periódico Indexado | Circulação/Nível: A - 1 |
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