Taq polymerase

DNA fragments were cloned by ligation cycle reaction (LCR) (66 (link)). Prior to electroporation, LCRs were precipitated with Pellet Paint (Novagen). For cloning purposes or screen purposes, we used Phusion Hot Start polymerase II (Fermentas) or Taq polymerase (Denville Scientific), respectively. For standard ligations, we used T4 DNA ligase (Thermo Scientific). Oligonucleotides and synthetic double-stranded DNA fragments were synthesized by Integrated DNA Technologies (IDT), and are listed in Table 5.

Cloning was performed via ligase cycling reaction (LCR) (24 (link)). Enzymes and reagents for LCR were purchased from Lucigen. PCR for cloning purposes was performed with the high-fidelity enzyme Phusion Hot Start polymerase II (Fermentas). PCR for screening purposes was performed with Taq polymerase (Denville Scientific). To concentrate the LCR before electrotransformation into L. reuteri, we used Pellet Paint coprecipitant (Novagen). Oligonucleotides and synthetic double-stranded DNA fragments were purchased from Integrated DNA Technologies (IDT). All oligonucleotides and synthetic DNA fragments used in this study are listed in Table S2.