All plasmids are listed in Supporting Information Table S2 . The primers designed for heterologous expression of the genes in A. nidulans are listed in Table S3 . Plasmid preparation, digestion with restriction enzymes, and gel electrophoresis were performed by standard methods. The coexpression strategy of the xen genes involved the construction of the plasmids for heterologous expression in A. nidulans is shown in Supporting Information Fig. S3A . Yeast assembly approach was used as described previously29 (link). The pWY25.16, pYWL27 and pYWB2 plasmids were used to construct the expression vectors, as shown in Table S2 . The gDNA fragments containing the xenA-xenG genes were amplified from gDNA of X. sinensis ML-31 using Phusion® high-fidelity DNA polymerase and the corresponding primers, as shown in Table S3 . The transformation of S. cerevisiae BJ5464-NpgA was carried out according to the manufacturer's protocol for an S.c.EasyComp transformation kit (Invitrogen). Yeast colonies were screened by PCR, and the plasmids were isolated using a Zymoprep kit (D2001, Zymo Research). The yeast plasmids were transformed into E. coli DH5α to obtain the plasmids used for verification and subsequent transformation into A. nidulans.
Zymoprep kit
Manufactured by Zymo Research
Sourced in United States
The Zymoprep kit is a DNA extraction and purification solution developed by Zymo Research. It is designed to efficiently isolate and purify DNA from a variety of biological samples. The kit utilizes a proprietary technology to capture and recover DNA, while removing contaminants and inhibitors. It provides a simple and effective method for obtaining high-quality DNA for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
S c easycomp transformation kit, by Thermo Fisher Scientific (1 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
8 oxo 2 dgtp, by TriLink (1 mentions)
S3e fluorescence activated cell sorter, by Bio-Rad (1 mentions)
Miseq adapter, by Illumina (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
6 protocols using zymoprep kit
1
Heterologous Expression of Xenian Genes
2
Isolating and Sequencing Yeast Plasmid DNA
3
Yeast Display Library Generation of scVEGF Variants
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The scVEGF constructs were prepared as described27 and cloned into the pCT yeast display plasmid. Library DNA containing random mutations was generated from scVEGFmut using error‐prone PCR and homologous recombination as described previously.28 , 44 Briefly, a range of mutation frequencies (∼0.2–2%) was obtained using Taq polymerase (Invitrogen) and varying amounts of the nucleotide analogs 8‐oxo‐2′‐dGTP and 2′‐dPTP (TriLink Biotech) in separate PCR reactions consisting of 5 cycles (200 µM analogs), 10 cycles (2 or 20 µM analogs), or 20 cycles (2 µM analogs). PCR products were amplified in the absence of analogs, and a total of 10 μg of mutated cDNA insert and 1 μg restriction‐digested pCT backbone were transformed into EBY100 yeast by electroporation. To create a second‐generation library, plasmid DNA was extracted from yeast after rounds 5 and 6 of library sorting using a ZymoPrep kit (Zymo Research), and subjected to error‐prone PCR and yeast transformation as described above. In both cases, library sizes of ∼0.5–2 × 107 transformants were obtained, as estimated by plating serial dilutions on selective media and colony counting.
4
Yeast-displayed scFv library screening for IL-17A binders
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Yeast-displayed first-generation and affinity maturation scFv libraries were grown in a SD-CAA selective medium and induced for expression with 2% w/v galactose at 30 °C overnight as described in Chao G et al.36 (link). The library was incubated with 500 nM to 10 nM biotinylated human IL-17A in PBS 0.1% BSA for 1 h, then washed three times with PBS 0.1% BSA and labeled with fluorescent-labeled antibodies mouse anti-Myc-FITC (Miltenyi Biotec, USA, cat:130-116-485) streptavidin APC (Jackson Immunoresearch, USA, cat 016-130-084). Post labeling the library was sorted on either BD ARIA III or BioRad S3e Fluorescence Activated Cell Sorter for high-affinity binders of human IL-17A. Gating strategy: EBY-100 cells labeled for FITC fluorescence (expression) above null control, which also showed top 1% APC fluorescence (binding) were considered as best binders and gated for collection. Isolated clones from the final sort were sequenced by extraction of plasmid DNA from the yeast clones using a Zymoprep kit (Zymo Research, USA) and the DNA was sequenced.
For the affinity maturation screen, the library was screened in the same fashion, but the yeast was induced at 37 °C to 40 °C and labeled with 1 nM–100 nM biotinylated IL-17A.
For the affinity maturation screen, the library was screened in the same fashion, but the yeast was induced at 37 °C to 40 °C and labeled with 1 nM–100 nM biotinylated IL-17A.
5
Illumina-based Sequence Analysis of Plasmid Libraries
6
High-Throughput Yeast-Based TAPBPR Mutagenesis
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Generation of the TAPBPR library and yeast preparation are described above. Naive and sorted yeast cultures were lysed with 125 U/ml Zymolase (37 °C, 5 h) and plasmid DNA was purified using a Zymoprep kit (Zymo Research). The mutated region of TAPBPR was PCR amplified in two stages. A first round of PCR used primers that added sequences complementary for Illumina sequencing primers (Supplementary Table 2 ). A second round of PCR added end sequences for annealing to the Illumina flow cell and included 6 bp barcodes for unique sample identification. Amplicons were sequenced on a Illumina HiSeq 4000 and data were analyzed with Enrich49 (link). Scripts for analysis are included in the GEO submission.
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