Hifi cloning

The pACYC184-CHL harboring a CHL resistance gene (cat, which encodes a CHL acetyltransferase) was generated from pACYC184 as template (GenBank X06403) using HiFi cloning (New England BioLabs, Ipswich, MA, USA) and PCR. The PCR fragment for cloning was amplified with primers listed in Table S3 using Q5 DNA polymerase (NEB). The purified HiFi cloning product (pACYC184-CHL) was used to transform electrocompetent E. coli DH5α. The recombinant plasmid was extracted using GeneJET Plasmid Miniprep (Thermo Fisher Scientific, Roskilde, Denmark) and confirmed by Sanger sequencing (Macrogen Europe). Then, MG1655/pTF2 and mutants were transformed with the recombinant plasmid using electroporation.

Oligonucleotides were purchased from Sigma Aldrich. Other materials purchased from Fisher Scientific, New England Biolabs, or as specified. Batches of >97% purity DDD01510706 (see Supplemental Figure 1) were prepared at the University of Dundee as previously described (Baragana et al., 2019 (link)). DDD01510706 is available upon request from Dr. Pawlowic (https://mrcppureagents.dundee.ac.uk/). Wild type Cryptosporidium parvum (Iowa II strain) oocysts were purchased from Bunchgrass Farms (Idaho, USA). Paromomycin sulfate and trimethoprim (TMP) (catalog #FT47738) were purchase from Carbosynth. Plasmids were constructed using HiFi cloning (NEB) and DNA sequence confirmed by Sanger sequencing (Genewiz). mScarlet-I was codon-optimized for Cryptosporidium parvum, produced as a gBlock by Integrated DNA Technologies.

For functional gene analysis, chicken embryos were injected and electroporated with long dsRNA (300 ng/µl) derived from the target gene (500 ng/µl for dsCables1) and a plasmid encoding β-actin-driven hrGFP (25 ng/µl). For hypomorphic experiments, combination of low doses (75 ng/µl) of each dsRNA were used (see Table S1 for details).
For the pY489 phospho-mutant versions of β-catenin, we used the Q5 Site-directed mutagenesis kit (New England Biolabs) to generate β-cateninY489F, a form of β-catenin that cannot be phosphorylated at tyrosine 489 due to the exchange of tyrosine 489 with phenylalanine, and β-cateninY489E (exchange of tyrosine 489 with glutamic acid), a phosphomimetic form of β-catenin that is dominant active. The following primers were used for the phosphomimetic substitution of Tyr (tat) by Glu (gaa): Fw, 5′-TCGCCTTCATcaaGGACTGGCCTGTTG-3′; Rv, 5′-ACGGCATTCTGGGCCATC-3′. For the phosphoinhibited substitution of Tyr (tat) by Phe (ttt): Fw, 5′-TCGCCTTCATtttGGACTGCCTG-3′; Rv, 5′-ACGGCATTCTGGGCCATC-3′. For rescue experiments, the open reading frame of mouse Cables1 was obtained from Biocat/Origene. The amplified PCR fragment was subcloned via HIFI cloning (New England Biolabs) under a Math1 enhancer.