Constructing Cell-Free Expression Systems for Low-Cost Access

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dc.contributor.authorGuzman-Chavez, Fernando
dc.contributor.authorArce, Anibal
dc.contributor.authorAdhikari, Abhinav
dc.contributor.authorVadhin, Sandra
dc.contributor.authorAntonio Pedroza-Garcia, Jose
dc.contributor.authorGandini, Chiara
dc.contributor.authorAjioka, Jim W.
dc.contributor.authorMolloy, Jenny
dc.contributor.authorSanchez-Nieto, Sobeida
dc.contributor.authorVarner, Jeffrey D.
dc.contributor.authorFederici, Fernan
dc.contributor.authorHaseloff, Jim
dc.date.accessioned2025-01-20T21:10:04Z
dc.date.available2025-01-20T21:10:04Z
dc.date.issued2022
dc.description.abstractCell-free systems for gene expression have gained attention as platforms for the facile study of genetic circuits and as highly effective tools for teaching. Despite recent progress, the technology remains inaccessible for many in low- and middle-income countries due to the expensive reagents required for its manufacturing, as well as specialized equipment required for distribution and storage. To address these challenges, we deconstructed processes required for cell-free mixture preparation and developed a set of alternative low-cost strategies for easy production and sharing of extracts. First, we explored the stability of cell-free reactions dried through a low-cost device based on silica beads, as an alternative to commercial automated freeze dryers. Second, we report the positive effect of lactose as an additive for increasing protein synthesis in maltodextrin-based cell-free reactions using either circular or linear DNA templates. The modifications were used to produce active amounts of two high-value reagents: the isothermal polymerase Bst and the restriction enzyme BsaI. Third, we demonstrated the endogenous regeneration of nucleoside triphosphates and synthesis of pyruvate in cell-free systems (CFSs) based on phosphoenol pyruvate (PEP) and maltodextrin (MDX). We exploited this novel finding to demonstrate the use of a cell-free mixture completely free of any exogenous nucleotide triphosphates (NTPs) to generate high yields of sfGFP expression. Together, these modifications can produce desiccated extracts that are 203-424-fold cheaper than commercial versions. These improvements will facilitate wider use of CFS for research and education purposes.
dc.fuente.origenWOS
dc.identifier.doi10.1021/acssynbio.1c00342
dc.identifier.issn2161-5063
dc.identifier.urihttps://doi.org/10.1021/acssynbio.1c00342
dc.identifier.urihttps://repositorio.uc.cl/handle/11534/93561
dc.identifier.wosidWOS:000776274500010
dc.issue.numero3
dc.language.isoen
dc.pagina.final1128
dc.pagina.inicio1114
dc.revistaAcs synthetic biology
dc.rightsacceso restringido
dc.subjectcell-free protein synthesis
dc.subjectlyophilization
dc.subjectNTPs
dc.subjectlactose
dc.subjectlow-cost
dc.subjectmaltodextrin
dc.subject.ods03 Good Health and Well-being
dc.subject.odspa03 Salud y bienestar
dc.titleConstructing Cell-Free Expression Systems for Low-Cost Access
dc.typeartículo
dc.volumen11
sipa.indexWOS
sipa.trazabilidadWOS;2025-01-12
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