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Filamentation and biofilm formation are regulated by the phase-separation capacity of network transcription factors in Candida albicans [1]

['Collin Ganser', 'Molecular Microbiology', 'Immunology Department', 'Brown University', 'Providence', 'Rhode Island', 'United States Of America', 'Mae I. Staples', 'Maureen Dowell', 'Corey Frazer']

Date: 2023-12

Plasmid construction

C. albicans TF constructs were created in pSFS2A [80] or derivatives of this plasmid as indicated below. A plasmid containing full length Efg1, pRB360, was constructed as described [81]. The Efg1 ΔN PrLD plasmid (pRB630) was created via fusion PCR of two fragments amplified from pRB360: (1) Efg1 5’ flank with oligos 1838/3916; and (2) Efg1 ΔN PrLD ORF with oligos 3915/1839. Splicing of the two fragments with overlap extension (SOE)-PCR was conducted with oligos 1838/1839, and the resulting fragment cloned into pSFS2A with ApaI/KpnI [82]. The Efg1 ΔC PrLD plasmid (pRB632) was created with fusion PCR of two fragments amplified from pRB360: (1) Efg1 5’ flank and ΔC ORF with oligos 1838/3918; and (2) remaining Efg1 ΔC ORF and 3’ flank with oligos 3917/1839. Fragments were fused with SOE-PCR and the product cloned into pSFS2A with ApaI/KpnI. The Efg1 ΔNC PrLD plasmid (pRB634) was created with fusion PCR of three fragments amplified from pRB360: (1) Efg1 5’ flank with oligos 1838/3916; (2) Efg1 DNA binding domain with oligos 3915/3918; and (3) Efg1 3’ flank with oligos 3917/1839. Fragments were fused via SOE-PCR and cloned into pSFS2A using ApaI/KpnI.

For Efg1 PrLD amino acid mutants, PrLD sequences were synthesized by BioBasic, and plasmids assembled via Golden Gate Assembly (GGA). A Golden Gate Assembly (GGA)-adapted version of pSFS2A (pRB1397) was generated by annealing oligos 6048/6049 to create a short double-stranded DNA fragment containing two outward facing BsaI sites, and sticky ends allowing ligation into pSFS2A digested with ApaI/XhoI. The Efg1 3’ flank sequence was amplified from C. albicans SC5314 gDNA with oligos 6422/6423 and cloned into pRB1397 with SacII/SacI to generate pRB1763. The resulting plasmid was then used as the destination vector for all Efg1 PrLD amino acid mutant GGA reactions. The Efg1 YF-to-S PrLD construct (pRB1610) was created via GGA of four PCR fragments: (1) Efg1 5’ flank from gDNA with oligos 6376/6377; (2) Efg1 N-terminal YF-to-S PrLD from pRB1858 with oligos 6378/6379; (3) Efg1 DNA binding domain from gDNA with oligos 6380/6381; and (4) Efg1 C-terminal YF-to-S PrLD from pRB1858 with oligos 6382/6383. Fragments were assembled via GGA with BsaI-HFv2 (NEB). The Efg1 ΔpolyQ PrLD construct (pRB1612) was created with GGA of four PCR fragments: (1) Efg1 5’ flank from gDNA with oligos 6376/6391; (2) Efg1 N-terminal ΔpolyQ PrLD from pRB1860 with oligos 6392/6393; (3) Efg1 DNA binding domain from gDNA with oligos 6394/6395; and (4) Efg1 C-terminal ΔpolyQ PrLD from pRB1860 with oligos 6396/6397. Fragments were assembled by GGA reaction with BsaI-HFv2. The Efg1 polyQG PrLD construct (pRB1611) was created via GGA of four PCR fragments: (1) Efg1 5’ flank from gDNA with oligos 6376/6384; (2) Efg1 N-terminal polyQG PrLD from pRB1859 with oligos 6385/6386; (3) Efg1 DNA binding domain from gDNA with oligos 6387/6388; and (4) Efg1 C-terminal polyQG PrLD from pRB1859 with oligos 6389/6390. Fragments were assembled by GGA with BsaI-HFv2. The Efg1-Taf15 plasmid (pRB1946) was created via GGA with 4 pieces: (1) Efg1 5’ flank was amplified from gDNA with oligos 6376/6377; (2) Taf15 PrLD was amplified from pRB1210 with oligos 7771/7772; (3) The Efg1 DBD was amplified from gDNA with oligos 7775/7776; (4) Taf15 IDR was amplified from pRB1210 with oligos 7773/7774. Fragments were assembled into the vector by GGA with BsaI-HFv2.

Brg1 and PrLD mutant derivatives were made via PCR and cloned into pSFS2A. The backbone for all of these constructs included the Brg1 3’ flank, amplified from gDNA with oligos 6103/6104, then cloned into pSFS2A with SacII/SacI. For the full-length construct (pRB1601), the BRG1 ORF (with 5’ flank) was amplified from gDNA with oligos 6099/6102, then cloned into the pSFS2A vector with KpnI/ApaI. For the Brg1 ΔN PrLD plasmid (pRB1602), two fragments were amplified from gDNA: (1) 5’ flank with ORF (no N-terminal PrLD) with oligos 6099/6113; and (2) remainder of the ORF with oligos 6112/6102. SOE-PCR was used to fuse fragments together, and the resulting product was digested with KpnI/ApaI and cloned into pSFS2A. For the Brg1 ΔC PrLD plasmid (pRB1603), the ORF without the C-terminal PrLD was amplified from gDNA with oligos 6099/6105, and the fragment cloned into the pSFS2A vector with KpnI/ApaI. The Brg1 ΔNC PrLD plasmid (pRB1604) was created via fusion PCR of two fragments: (1) 5’ flank with ORF (no N-terminal PrLD) using oligos 6099/6113; and (2) remainder of ORF (no C-terminal PrLD) with oligos 6112/6105. Fragments were stitched together via SOE-PCR, and the product cloned into pSFS2A with ApaI/KpnI.

For the Brg1 PrLD mutants, PrLD sequences were synthesized by BioBasic and plasmids assembled via GGA using the modified pSFS2A plasmid with the Brg1 3’ flank, which was amplified from gDNA with oligos 6103/6104 and cloned into pRB1397 with SacII/SacI. The Brg1 YF-to-S PrLD plasmid (pRB1739) was made via GGA of four fragments: (1) Brg1 5’ flank amplified from gDNA with oligos 6569/6570; (2) YF-to-S N-terminal PrLD amplified from pRB1862 with oligos 6571/6572; (3) Brg1 DNA binding domain amplified from gDNA with oligos 6573/6574; and (4) YF-to-S C-terminal PrLD amplified from pRB1862 with oligos 6575/6576. Fragments were assembled via GGA with BsaI-HFv2. The Brg1 ΔpolyQ PrLD plasmid (pRB1740) was made via GGA of four fragments: (1) Brg1 5’ flank amplified from gDNA with oligos 6569/6570; (2) ΔpolyQ N-terminal PrLD amplified from pRB1864 with oligos 6571/6572; (3) Brg1 DNA binding domain amplified from gDNA with oligos 6573/6614; and (4) ΔpolyQ C-terminal PrLD amplified from pRB1864 with oligos 6615/6583. Fragments were assembled by GGA with BsaI-HFv2. The Brg1 polyQG PrLD plasmid (pRB1741) was made via GGA of four fragments: (1) Brg1 5’ flank amplified from gDNA with oligos 6569/6570; (2) polyQG N-terminal PrLD amplified from pRB1863 with oligos 6571/6577; (3) Brg1 DNA binding domain amplified from gDNA with oligos 6573/6578; and (4) polyQG C-terminal PrLD amplified from pRB1863 with oligos 6579/6580. Fragments were assembled by GGA with BsaI-HFv2.

All Bcr1 plasmids were constructed by GGA. The backbone plasmid for the wildtype gene and PrLD mutants was the modified pSFS2A GGA plasmid with the Bcr1 3’ flank sequence, which was amplified from gDNA with oligos 6095/6096 and cloned into pRB1397 with SacII/SacI. For the full length Bcr1 plasmid (pRB1742), the BCR1 ORF (with 5’ flank) was amplified from gDNA with oligos 6622/6625 and the resulting product assembled by GGA with BsaI-HFv2. The Bcr1 ΔN PrLD plasmid (pRB1743) was assembled from two PCR fragments: (1) Bcr1 5’ flank amplified from gDNA with oligos 6622/6623; and (2) the BCR1 ORF (no N-terminal PrLD) amplified from gDNA with oligos 6624/6625. Fragments were assembled by GGA reaction with BsaI-HFv2. The Bcr1 ΔC PrLD plasmid (pRB1744) was assembled from two PCR fragments: (1) Bcr1 5’ flank amplified from gDNA with oligos 6626/6627; and (2) the BCR1 ORF (no C-terminal PrLD) was amplified from gDNA with oligos 6628/6629. Fragments were assembled by GGA with BsaI-HFv2. The Bcr1 ΔNC PrLD plasmid (pRB1745) was assembled from three PCR fragments: (1) Bcr1 5’ flank amplified from gDNA with oligos 6630/6631; (2) the BCR1 ORF (no N-terminal PrLD) amplified from gDNA with oligos 6632/6633; and (3) the BCR1 ORF (no C-terminal PrLD) amplified from gDNA with oligos 6634/6635. Fragments were assembled by GGA with BsaI-HFv2.

For the Bcr1 PrLD amino acid mutant plasmids, PrLD sequences were synthesized by BioBasic. The Bcr1 YF-to-S PrLD plasmid (pRB1746) was made via GGA of five fragments: (1) Bcr1 5’ flank amplified from gDNA with oligos 6863/6864; (2) YF-to-S N-terminal PrLD amplified from pRB1865 with oligos 6865/6866; (3) Bcr1 DNA binding domain amplified from gDNA with oligos 6867/6868; (4) YF-to-S C-terminal PrLD amplified from pRB1865 with oligos 6869/6870; and (5) the remaining BCR1 ORF amplified from gDNA with oligos 6871/6872. Fragments were assembled by GGA with BsaI-HFv2. The Bcr1 ΔpolyQ PrLD plasmid (pRB1747) was made via GGA of five fragments: (1) Bcr1 5’ flank amplified from gDNA with oligos 6873/6874; (2) ΔpolyQ N-terminal PrLD amplified from pRB1864 with oligos 6875/6876; (3) Bcr1 DNA binding domain amplified from gDNA with oligos 6877/6878; (4) ΔpolyQ C-terminal PrLD amplified from pRB1864 with oligos 6879/6880; and (5) the remaining BCR1 ORF amplified from gDNA with oligos 6881/6882. Fragments were assembled by GGA with BsaI-HFv2. The Bcr1 polyQG PrLD plasmid (pRB1748) was made via GGA of five fragments: (1) Bcr1 5’ flank amplified from gDNA with oligos 6883/6884; (2) polyQG N-terminal PrLD amplified from pRB1866 with oligos 6885/6886; (3) Bcr1 DNA binding domain amplified from gDNA with oligos 6887/6888; (4) ΔpolyQ C-terminal PrLD amplified from pRB1866 with oligos 6889/6890; and (5) the remaining BCR1 ORF amplified from gDNA with oligos 6891/6892. Fragments were assembled by GGA with BsaI-HFv2.

All Flo8 constructs were created via PCR and cloned into the pSFS2A backbone. The backbone included the Flo8 3’ flank, amplified from gDNA with oligos 6089/6090, then cloned into pSFS2A with SacII/SacI. For the full length Flo8 plasmid (pRB1790), the FLO8 ORF (with 5’ flank) was amplified from gDNA with oligos 6085/6088, and the product cloned into pSFS2A with ApaI/XhoI. The Flo8 ΔN PrLD DNA sequence was synthesized by Twist BioScience. The Flo8 ΔN PrLD plasmid (pRB1791) was made via fusion PCR of two fragments: (1) Flo8 5’ flank amplified from gDNA with oligos 6085/6086; and (2) Flo8 ΔN PrLD amplified from pRB1871 with oligos 6108/6088. The products were fused together with SOE-PCR and the fragment cloned into the pSFS2A vector with ApaI/XhoI. The Flo8 PrLD amino acid mutants were synthesized by BioMatik. The Flo8 YF-to-S PrLD plasmid (pRB1793) was made via PCR, with the YF-to-S PrLD (including 5’ flank) amplified from pRB1867 with oligos 6085/6088, then cloned into the pSFS2A vector with ApaI/XhoI. The Flo8 ΔpolyQ PrLD plasmid (pRB1794) was made via PCR, with the ΔpolyQ PrLD (including 5’ flank) amplified from pRB1868 with oligos 6085/6088, then cloned into the pSFS2A vector with ApaI/XhoI. The Flo8 polyQG PrLD plasmid (pRB1795) was made via PCR, with the polyQG PrLD (including 5’ flank) amplified from pRB1869 with oligos 6085/6088, then cloned into the pSFS2A vector with ApaI/XhoI. The Flo8-Taf15 plasmid (pRB2042) was assembled with GGA with 3 pieces using the pSFS2A vector with Flo8 3’ flank as the backbone. (1) Flo8 5’ flank and N terminal region were amplified from gDNA with oligos 7777/7780; (2) Taf15 IDR was amplified from pRB1210 with oligos 7783/7772; (3) Flo8 C terminal region was amplified from gDNA with oligos 7781/7782. Fragments were assembled by GGA with BsaI-HFv2.

For protein assays, EFG1 and FLO8 ORFs were codon-optimized for expression in E. coli. The synthetic ORFs were then cloned into plasmid pRP1B–MBP/THMT (pRB523) with restriction enzymes NdeI/XhoI to create plasmids pRB514 and pRB971, respectively [83, 84]. The GFP-CTD of RNA Pol II was created via fusion PCR of 2 fragments: (1) GFP was amplified from pRB690 with oligos 4877/4878; and (2) the C-terminal domain of RNA Pol II was amplified from pRB984 (codon-optimized for E. coli expression, synthesized by Biomatik) with oligos 5084/5085. SOE PCR was carried out to fuse the two fragments with oligos 4877/5085. The resulting product was cloned into pRB523 with restriction enzymes NheI/XhoI to generate pRB1034. The pMBP–GFP plasmid (pRB723) was created by PCR amplifying GFP from pRB690 (oligos 4122/4123), which was cloned into pRB523 with NheI/XhoI.

For expression of C. albicans TF PrLDs in U2OS LacO cells, as either LacI-EYFP or mCherry fusions, plasmids were constructed with codon-optimized sequences for expression in E. coli. The Efg1-PrLD-LacI-EYFP plasmid (pRB1222) was constructed previously [28]. The Flo8-PrLD-LacI-EYFP plasmid (pRB1262) was constructed by amplifying the FLO8 PrLD from pRB960 using oligos 5680/5681. The resulting insert was digested and cloned into pRB1208 with BsrGI/BspEI. The Brg1-PrLD-LacI-EYFP (pRB1595) construct was created via a fusion PCR of three fragments: (1) the N-terminal PrLD of Brg1 was amplified from pRB832 with oligos 6502/6503; (2) EYFP was amplified from pRB1208 with oligos 6518/6519; (3) the C-terminal PrLD of Brg1 was amplified from pRB832 with oligos 6504/6505. SOE PCR was carried out with oligos 6502/6505. The fragment was digested with SpeI/BspEI and cloned into pRB1208 digested with NheI/BspEI (SpeI and NheI yield compatible sticky ends for ligation reaction). The Bcr1-PrLD-LacI-EYFP plasmid (pRB1597) was created via a three-way fusion PCR: (1) the Bcr1 N-terminal PrLD was amplified from pRB841 with oligos 6510/6511; (2) EYFP was amplified from pRB1208 with oligos 6518/6519; (3) the Bcr1 C-terminal PrLD was amplified from pRB841 with oligos 6512/6513. SOE PCR with oligos 6510/6513 yielded a fusion product that was digested with NheI/BspEI and cloned into pRB1208.

The Efg1 YF-to-S PrLD-LacI-EYFP plasmid (pRB2046) was created by GGA with pSFS2a using: (1) Efg1 YF-to-S N PrLD amplified from pRB1789 with oligos 7897/7898; (2) EYFP amplified from pRB1222 with oligos 7899/7825; (3) Efg1 YF-to-S C PrLD amplified from pRB1789 with oligos 7826/7900. Fragments were assembled by GGA with BsaI-HFv2. The cassette was amplified by PCR with oligos 8030/8031, digested with BspEI and NheI-HF and cloned into pRB1208. The Brg1 YF-to-S PrLD-LacI-EYFP plasmid (pRB2045) was created by GGA with pSFS2a using: (1) Brg1 YF-to-S N PrLD amplified from pRB1785 with oligos 7746/7747; (2) EYFP amplified from pRB1222 with oligos 7744/7745; (3) Brg1 YF-to-S C PrLD amplified from pRB1785 with oligos 7806/7807. Fragments were assembled by GGA with BsaI-HFv2. The cassette was amplified by PCR with oligos 8028/8029, digested with AgeI and BspEI and cloned into pRB1208.

The Efg1-PrLD-mCherry plasmid (pRB1224) was constructed as described [28]. The Flo8-PrLD-mCherry plasmid (pRB1264) was created via PCR amplification of the Flo8 PrLD from pRB960 using oligos 5680/5682. The resulting product was cloned into pRB1207 with BsrGI/BspEI.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1011833

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