The genus Streptomyces are valuable producers of antibiotics and other pharmaceutically important bioactive compounds.Advances in molecular engineering tools,such as CRISPR,have provided some access to the metabolic p...The genus Streptomyces are valuable producers of antibiotics and other pharmaceutically important bioactive compounds.Advances in molecular engineering tools,such as CRISPR,have provided some access to the metabolic potential of Streptomyces,but efficient genetic engineering of strains is hindered by laborious and slow manual transformation protocols.In this paper,we present a semi-automated medium-throughput workflow for the introduction of recombinant DNA into Streptomyces spp.using the affordable and open-sourced Opentrons(OT-2)robotics platform.To increase the accessibility of the workflow we provide an open-source protocol-creator,ActinoMation.ActinoMation is a literate programming environment using Python in Jupyter Notebook.We validated the method by transforming Streptomyces coelicolor(M1152 and M1146),S.albidoflavus(J1047),and S.venezuelae(DSM40230)with the plasmids pSETGUS and pIJ12551.We demonstrate conjugation efficiencies of 3.33*10^(-3)/0.33%for M1152 with pSETGUS and pIJ12551;2.96*10^(-3)/0.29%for M1146 with pSETGUS and pIJ12551;1.21*10^(-5)/0.0012%for J1047 with pSETGUS and 4.70*10^(-4)/0.047%with pIJ12551,and 4.97*10^(-2)/4.97%for DSM40230 with pSETGUS and 6.13*10^(-2)/6.13%with pIJ12551 with a false positive rate between 8.33%and 54.54%.Automation of the conjugation workflow facilitates a streamlined workflow on a larger scale without any evident loss of conjugation efficiency.展开更多
基金funded by grants from the Novo Nordisk Foundation(NNF20CC0035580,NNF22OC0078997).
文摘The genus Streptomyces are valuable producers of antibiotics and other pharmaceutically important bioactive compounds.Advances in molecular engineering tools,such as CRISPR,have provided some access to the metabolic potential of Streptomyces,but efficient genetic engineering of strains is hindered by laborious and slow manual transformation protocols.In this paper,we present a semi-automated medium-throughput workflow for the introduction of recombinant DNA into Streptomyces spp.using the affordable and open-sourced Opentrons(OT-2)robotics platform.To increase the accessibility of the workflow we provide an open-source protocol-creator,ActinoMation.ActinoMation is a literate programming environment using Python in Jupyter Notebook.We validated the method by transforming Streptomyces coelicolor(M1152 and M1146),S.albidoflavus(J1047),and S.venezuelae(DSM40230)with the plasmids pSETGUS and pIJ12551.We demonstrate conjugation efficiencies of 3.33*10^(-3)/0.33%for M1152 with pSETGUS and pIJ12551;2.96*10^(-3)/0.29%for M1146 with pSETGUS and pIJ12551;1.21*10^(-5)/0.0012%for J1047 with pSETGUS and 4.70*10^(-4)/0.047%with pIJ12551,and 4.97*10^(-2)/4.97%for DSM40230 with pSETGUS and 6.13*10^(-2)/6.13%with pIJ12551 with a false positive rate between 8.33%and 54.54%.Automation of the conjugation workflow facilitates a streamlined workflow on a larger scale without any evident loss of conjugation efficiency.
