Bead-Based Assay for Spatiotemporal Gene Expression Control in Cell-Free Transcription–translation Systems

Abstract

Cell-free gene expression has applications in synthetic biology, biotechnology and biomedicine. In this technique gene expression regulation plays an important role. Transcription factors do not completely suppress expression while other methods for expression control, for example CRISPR/Cas, often require important biochemical modifications. Here we use an all Escherichia coli-based cell-free expression system and present a bead-based method to instantly start and, at a later stage, completely stop gene expression. Magnetic beads coated with DNA of the gene of interest trigger gene expression. The expression stops if we remove the bead-bound DNA as well as transcribed mRNA by hybridization to bead-bound ssDNA. Our method is a simple way to control expression duration very accurately in time and space.

A bead-based method for the removal of the amino acid lysine from cell-free transcription-translation systems

“…This plasmid is optimized for our cell-free system. It can be derived from the commercially available plasmid pBest-Or2-Or1-Pr-UTR1-deGFP-T500 (Addgene Cat# 40019) (Finkler and Ott, 2019;Schenkelberger et al, 2017;Shin and Noireaux, 2010). The gene for the high-lysine dummy protein (HLDP), DNA-sequence in Supplementary Fig.…”

“…The gene coding for the fusion-protein GHLDP is linked to magnetic beads (Finkler and Ott, 2019;Invitrogen, 2017). Using the primers Biotin-TEG-Seq1 (TEG stands for TriEthyleneGlycol) (5′-Biotin-TEG-CAC CAT CAG CCA GAA AAC C-3′, Metabion) and Seq4 (5′-GAG CTG ACT GGG TTG AAG G-3′, Metabion) and streptavidin-coated magnetic beads T1 (Dynabeads® MyOne™ Streptavidin T1, Invitrogen) we obtain the construct T1-Biotin-TEG-Seq1-Or2-Or1-Pr-UTR1-deGFP-HLDP-T500-Seq4 (T1-GHLDP) at a DNA-concentration of 400 nM.…”

“…As previously shown, mRNA is quite stable in our system. It needs to be removed before new genes can be added (Finkler and Ott, 2019). For removal, DNA complementary to the mRNA of GHLDP is used.…”

“…For experiments 1.5 µl of the T1-GHLDP stock solution and 2.1 µl of double-distilled water were added to the 8.4 µl aliquots. This concentration is optimal for bead-bound DNA (Finkler and Ott, 2019). Aliquots filled up with double-distilled water to 12 µl were taken as a reference for comparison.…”

“…Therefore the amount of magnetic beads has to be adjusted for each stock. The amount of amino acids, magnesium glutamate and potassium glutamate must be adjusted for each stock for optimal cell-free protein synthesis (Finkler and Ott, 2019;Shin and Noireaux, 2010).…”