ActinoMation: A literate programming approach for medium-throughput robotic conjugation of Streptomyces spp

The genus <i>Streptomyces</i> 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 <i>Streptomyces</i>, 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 <i>Streptomyces</i> 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 <i>Streptomyces coelicolor</i> (M1152 and M1146), <i>S. albidoflavus</i> (J1047), and <i>S. venezuelae</i> (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.