Sanger dna sequencing

The codon-optimized bacterial expression plasmid pET28a(+)APP695 encoding for N-terminal 6xHis-tagged wildtype human APP695 was synthesized by Biomatik (Toronto, ON, Canada). To generate the plasmid pET28a(+)N-APP encoding for the N-terminal APP695 fragment N-APP (amino acids 1 to 267 of APP695), the amino acid at 268 was substituted to the stop codon TAA. The plasmid pET28a(+)E2 encoding for the APP695 fragment E2 (amino acids 268 to 612) was generated in two steps by deleting amino acids 1 to 267 and substituting amino acid 613 to the stop codon TAA. The third plasmid, pET28a(+)APP-C encoding for the C-terminal APP695 fragment APP-C (amino acids 494 to 695), was produced by deletion of amino acids 1 to 493. The primer sets used for site-directed mutagenesis by inverse PCR-based were: 5‘-AGCAACCGAATAAACCACCAGCATTG-3′ and 5‘-TCTTCATACGGTTCTTCTG-3′ for N-APP, 5‘-TCATCAGAAATAAGTTTTCTTTGCAGAAGATGTTGG-3′ and 5‘-TGCACTTCATAACCGCTATC-3′ as well as 5‘-CGTACCACCAGCATTGCAA-3′ and 5‘-GGATCCGCGACCCATTTG-3′ for E2, and 5′-GAACAGAATTATAGTGATGATGTGC-3′ and 5‘-GGATCCGCGACCCATTTG-3′ for APP-C (Biomers.net, Ulm, Germany). Successful introduction of deletions and mutations was confirmed by Sanger DNA sequencing (Eurofins Genomics, Munich, Germany).

The plasmids for expressing recombinant versions of ReoM, PrkA-KD and PrpC were prepared by first amplifying the corresponding genes (reoM, lmo1820 and lmo1821) from L. monocytogenes EGD-e genomic DNA using primer pairs Lmo1503F/Lmo1503R, PrkAF/PrkAR, and PrpCF/PrpCR, respectively. The PCR products were individually ligated between the NcoI and XhoI sites of pETM11 (Peränen et al., 1996 (link)). All mutagenesis was carried out using the Quikchange protocol and the correct sequence of each plasmid and mutant constructed was verified by Sanger DNA sequencing (Eurofins Genomics).

Deletion of and ompF was done using the λ-Red recombinase-mediated gene deletion method [37 (link)]. A PCR product harboring 50 bp end sequences homologous to ompF and a chloramphenicol resistance marker was amplified with plasmid pKD3 as template and primers ompF_up (5′-ATTGACGGCAGTGGCAGGTGTCATAAAAAAAACCATGAGGGTAATAAATAGTGTAGGCTGGAGCTGCTTC-3′) and ompF_down (5′-AAACAGGACCAAAGTCCTGTTTTTTCGGCATTTAACAAAGAGGTGTGCTAATGGGAATTAGCCATGGTCC-3′). The PCR product was transformed into the appropriate E. coli strain harboring the λ-Red recombinase expression plasmid pKD46, and subsequently transformants were selected on media plates containing chloramphenicol. Deletion of ompF was verified by amplifying the appropriate chromosomal region using primer pairs and ompF1 (5′-CACTTTCACGGTAGCGAAAC-3′)/ompF2 (5′-CATGACGAGGTTCCATTATGG-3′), and confirming by Sanger DNA sequencing (Eurofins).

The codon-optimized bacterial expression vector pET28a(+)tau441 encoding for N-terminal 6xHis-tagged human microtubule-associated protein tau (MAPT) isoform 4 (tau441) was synthesized by Biomatik (Kitchener, ON, Canada). Plasmids encoding for tau441 fragments (tau441^1-155 and tau441^243-441) and phosphorylation site mutants of full-length tau441 (tau441^{S68A+T69A+T71A}, tau441^{S198A+S199A+S202A+T205A}, tau441^{T212A+S214A+T217A+T220A}, tau441^S289A, tau441^{S409A+S412A+S413A+T414A+S416A} and tau441^S422A+T427A) were created by using inverse PCR and the PCR primer pairs as indicated in Supplementary Table S1 (see Figure 1). Subsequently, PCR products were ligated. To generate plasmid pET28a(+)tau441^156-242, Gibson Assembly^® was performed according to manufacturer’s instructions (New England Biolabs Inc., Ipswich, NY, United States). Sanger DNA sequencing (Eurofins Genomics, Munich, Germany) confirmed successful introduction of mutations.