The Saccharomyces cerevisiae tps1Δ and tps2Δ mutants were derived from the CEN.PK133-5D ura3-52 strain 28 (link) and have been described elsewhere 15 (link)62 (link). The tps1Δ strain was used as the host for transformation with the YCplac33 plasmid 63 (link) bearing TPS1 from various origin (Saccharomyces cerevisiae, Drosophila melanogaster, Arabidopsis thaliana, Escherichia coli, Aspergillus nidulans, Hansenula polymorpha, Pichia pastoris, Magnaporthe grisea, Yarrowia lipolytica, Kluyveromyces lactis, Candida albicans, Schizosaccharomyces pombe and Ralstonia solanacearum) under the control of the S. cerevisiae PGK1 promoter. The TPS1 coding sequence from these organisms was amplified by PCR using 5’ and 3’ primers listed in Table S1. The PCR fragments were cloned into the YCplac33 plasmid using the In-Fusion system (Clontech), except the ones from M. grisea and P. pastoris, for which exons were amplified and assembled by using the NEBuilder® HiFi DNA Assembly system (New England Biolabs). All plasmid constructs bearing the TPS1 homologues are listed in Table 3 and were verified by DNA sequencing. The Lithium acetate procedure described in 64 (link) was used to transform S. cerevisiae tps1Δ mutant with the different plasmids.
Nebuilder hifi dna assembly system
Manufactured by New England Biolabs
The NEBuilder HiFi DNA assembly system is a molecular biology tool that enables the seamless and efficient assembly of multiple DNA fragments into a single construct. It utilizes a proprietary enzyme mix to join DNA segments with high fidelity, simplifying the process of creating complex genetic constructs.
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Lab products found in correlation
In fusion system, by Takara Bio (1 mentions)
Nucleospin plasmid easypure kit, by Macherey-Nagel (1 mentions)
Primestar gxl, by Takara Bio (1 mentions)
Dneasy powersoil kit, by Qiagen (1 mentions)
Nucleospin tissue kit, by Macherey-Nagel (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Qiaamp dna blood kit, by Qiagen (1 mentions)
7 protocols using nebuilder hifi dna assembly system
1
Heterologous TPS1 Expression in S. cerevisiae tps1Δ
2
Plasmid Engineering for bamA Complementation
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Two E. coli -Acinetobacter shuttle plasmids, pARP3 and pARKM, were used to generate plasmids to complement the bamA gene. A DNA fragment containing the bamA gene and its ribosome-binding site was amplified by PCR using the primers BamA_F/BamA_R and the genomic DNA of Tol 5 as a template, followed by cloning into the EcoRI-XbaI site of pARP3 using the NEBuilder HiFi DNA assembly system (New England Biolabs, Ipswich, MA) to generate pPBADBamA. To obtain a xylR-Pu promoter cassette, PCR was performed using the primers XylR_F/XylR_R and PU_F/PU_R and the plasmid pWW0 derived from P. putida mt-2 as a DNA template. The amplified fragments and XhoI-EcoRI-digested pPBADBamA were assembled using the NEBuilder HiFi DNA assembly system to generate pARP3-XylR-PuBamA. A XhoI–XbaI-digested fragment containing xylR, the Pu promoter, and bamA was cloned at the same site as pARPKM to generate pPuBamA.
To generate pJQBamAKO, a suicide plasmid for the deletion of the bamA gene, DNA fragments containing the 1.5-kb upstream and 1.7-kb downstream regions of bamA were amplified by PCR using the primers BamA_1500_F/BamA_1500_R and BamA_1700_F/BamA_1700_R. The amplicons and BamHI-digested pJQ200sk were assembled using the NEBuilder HiFi DNA assembly system.
To generate pJQBamAKO, a suicide plasmid for the deletion of the bamA gene, DNA fragments containing the 1.5-kb upstream and 1.7-kb downstream regions of bamA were amplified by PCR using the primers BamA_1500_F/BamA_1500_R and BamA_1700_F/BamA_1700_R. The amplicons and BamHI-digested pJQ200sk were assembled using the NEBuilder HiFi DNA assembly system.
3
Plasmid Engineering for bamA Complementation
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Two E. coli -Acinetobacter shuttle plasmids, pARP3 and pARKM, were used to generate plasmids to complement the bamA gene. A DNA fragment containing the bamA gene and its ribosome-binding site was amplified by PCR using the primers BamA_F/BamA_R and the genomic DNA of Tol 5 as a template, followed by cloning into the EcoRI-XbaI site of pARP3 using the NEBuilder HiFi DNA assembly system (New England Biolabs, Ipswich, MA) to generate pPBADBamA. To obtain a xylR-Pu promoter cassette, PCR was performed using the primers XylR_F/XylR_R and PU_F/PU_R and the plasmid pWW0 derived from P. putida mt-2 as a DNA template. The amplified fragments and XhoI-EcoRI-digested pPBADBamA were assembled using the NEBuilder HiFi DNA assembly system to generate pARP3-XylR-PuBamA. A XhoI–XbaI-digested fragment containing xylR, the Pu promoter, and bamA was cloned at the same site as pARPKM to generate pPuBamA.
To generate pJQBamAKO, a suicide plasmid for the deletion of the bamA gene, DNA fragments containing the 1.5-kb upstream and 1.7-kb downstream regions of bamA were amplified by PCR using the primers BamA_1500_F/BamA_1500_R and BamA_1700_F/BamA_1700_R. The amplicons and BamHI-digested pJQ200sk were assembled using the NEBuilder HiFi DNA assembly system.
To generate pJQBamAKO, a suicide plasmid for the deletion of the bamA gene, DNA fragments containing the 1.5-kb upstream and 1.7-kb downstream regions of bamA were amplified by PCR using the primers BamA_1500_F/BamA_1500_R and BamA_1700_F/BamA_1700_R. The amplicons and BamHI-digested pJQ200sk were assembled using the NEBuilder HiFi DNA assembly system.
4
Plasmid and Genomic DNA Extraction
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Small plasmids were extracted by using NucleoSpin® Plasmid EasyPure Kit (Macherey-Nagel). The total DNA of each strain (donors, recipients, and transconjugants) was extracted by using NucleoSpin® Tissue Kit (Macherey-Nagel). DNAs from cow manure and the sorted cells in PBS were performed with DNeasy PowerSoil Kit (Qiagen, GmbH, Hilden, Germany). Polymerase chain reaction (PCR) was carried out on a T100TM thermal cycler (Bio-Rad, Hercules, CA, United States) with the primer sets and PrimeSTAR® GXL (Takara Bio) or KOD One PCR Master Mix (TOYOBO Inc.). Restriction enzymes (New England Biolabs or Takara Bio), the HiYieldTM Gel/PCR DNA fragments Extraction kit (RBC Bioscience, New Taipei City, Taiwan), NEBuilder Hifi DNA Assembly system (New England Biolabs, Ipswich, MA, United States), and competent E. coli DH5α cells (RBCBioscience) were employed for cloning of DNA fragments. The other procedures were performed according to standard methods (Sambrook and Russell, 2001 ).
5
Genetic manipulation of P. falciparum RON11
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Genomic DNA was isolated from P. falciparum NF54attB cultures using the QIAamp DNA blood kit (Qiagen). PCR products were inserted into the respective plasmids using the NEBuilder HiFi DNA Assembly system (NEB). All constructs used in this study were confirmed by sequencing. All primers used in this study are in Table 3.1 .
For generation of the plasmid pKD-RON11-Apt, sequences of approximately 500 bp of homology to the RON11 (Pf3D7_1463900) C-terminus and 3’UTR were amplified using primer pairs P3-P4 and P5-P6, respectively (Table 3.1 ). Amplicons were then inserted into pKD (Rajaram et al. 2020 (link)) digested by with AatII and AscI. For expression of a RON11 gRNA, oligo P7 was inserted into cut pUF1-Cas9 (Table 3.1 ).
For generation of the plasmid pKD-RON11-Apt, sequences of approximately 500 bp of homology to the RON11 (Pf3D7_1463900) C-terminus and 3’UTR were amplified using primer pairs P3-P4 and P5-P6, respectively (
6
Retroviral Expression of PBX Transcription Factors
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Complementary DNAs encoding mouse PBX1, PBX2, PBX3, or PBX4, each with a COOH-terminal HA epitope tag, were subcloned into pMX-puro (kindly provided by T. Kitamura, The University of Tokyo, Japan) with the use of a NEBuilder HiFi DNA Assembly system (New England Biolabs). The resulting vectors were introduced into Plat-E packaging cells by transfection in order to generate recombinant retroviruses. 3T3-L1 cells were infected with the retroviruses in the presence of polybrene (5 µg/ml) and were then cultured in the presence of puromycin (5 µg/ml) for selection.
7
Establishing Stable Cell Lines Using Plasmids
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All plasmids used in this study are listed in Supplemental Table S1 with Benchling links that provides the sequences and plasmid maps. pNATZA11 [National Bio-Resource Project (NBRP) plasmid FYP2875, provided by Dr. Watanabe (The University of Tokyo) and pFA6a-mNG-kan [NBRP plasmid FYP4997] 41 (link) were used as vector backbones to construct plasmids for the establishment of stable cell lines of the fission yeast S. pombe. pCSIIpuro-mNG-eDHFR(69K6) 21 was used as a PCR template and lentiviral vector for the establishment of a stable mammalian cell line. All plasmids were generated using standard cloning procedures and the NEBuilder HiFi DNA assembly system (New England Biolabs).
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