Protocol for Cryptosporidium parvum Plasmid Construction

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.

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Hanna J.C., Corpas-Lopez V., Seizova S., Colon B.L., Bacchetti R., Hall G.M., Sands E.M., Robinson L., Baragaña B., Wyllie S, & Pawlowic M.C. (2023). Mode of action studies confirm on-target engagement of lysyl-tRNA synthetase inhibitor and lead to new selection marker for Cryptosporidium. Frontiers in Cellular and Infection Microbiology, 13, 1236814.

Publication 2023

Oligonucleotides HiFi cloning Sanger sequencing

Codon Cryptosporidium parvum Dna sequence Oligonucleotides Oocysts Paromomycin sulfate Plasmids Strain Trimethoprim

Corresponding Organization : University of Dundee

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Variable analysis

independent variables

DDD01510706 batch preparation

dependent variables

Cryptosporidium parvum parasite growth/inhibition

control variables

Oligonucleotides from Sigma Aldrich
Other materials from Fisher Scientific, New England Biolabs, or as specified
Wild type Cryptosporidium parvum (Iowa II strain) oocysts from Bunchgrass Farms (Idaho, USA)
Paromomycin sulfate and trimethoprim (TMP) from Carbosynth
Plasmid construction using HiFi cloning (NEB) and DNA sequence confirmation by Sanger sequencing (Genewiz)
MScarlet-I codon-optimized for Cryptosporidium parvum, produced as a gBlock by Integrated DNA Technologies

positive controls

Not specified

negative controls

Not specified

Annotations

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