Biorad gene pulser xcell apparatus

SNP replacements in gene cpsK were performed by allelic exchange. PCR amplicons were generated using specific primers and cloned into plasmid pCR2.1 (ThermoFisher), extracted using EcoRI, and subcloned into the thermosensitive E. coli-S. suis shuttle vector pSET4s^{38 (link)} previously digested with EcoRI, giving rise to replacement vectors p4cpskG483T and p4cpskT483G. These vectors were then electroporated into recipient S. suis strains using a Biorad Gene Pulser Xcell apparatus (BioRad) under specific conditions (12.5 kV/cm, 200 Ω, and 25 μF). Isoallelic mutants were isolated as previously described³⁹ . Sanger sequencing confirmed adequate replacement of nucleotide 483 of cpsK genes. Whole-genome sequencing using Illumina MiSeq technology of all parental and mutant strains, and polymorphism identification were performed as previously described^{21 (link)}.

The full-length cDNA of PtME from P. tricornutum cloned into the expression vector pHY11 previously was employed for transformation in this study. In this expression vector pHY11, PtME was cloned under the control of fcpC promoter and fcpA terminator from fucoxanthin chlorophyll a/c binding protein genes of P. tricornutum. C. pyrenoidosa was electroporated with the expression vector using Bio-Rad GenePulserXcell apparatus (Bio-Rad, USA) as previously reported [11 (link)]. The electroporated algal cells were transferred into liquid medium and cultured at shaker at dark for 24 h. Afterwards, the transformed cells were plated onto selection medium fortified with 10 μg ml⁻¹ zeocin. The growing transformed cells in the selection medium were cultured in liquid medium with chloramphenicol and subcultured every week.

The pPha-VCP vector containing the bidirectional VCP2 promoter (from N. oceanica) and VCP1 3’UTR terminator (from N. oceanica) as well as sh ble resistance gene was constructed^{22 (link), 36 (link)}. The enhanced green fluorescent protein (eGFP) fragment was amplified by PCR from the pPhaT1-eGFP vector^{36 (link)} using the primers eGFP_ KpnI_f and eGFP_XbaI_r and ligated into the pPha-VCP vector (between the KpnI and XbaI sites), and was named as pPha-VCP-eGFP. For the localization analysis of PDK, a construct was generated to express C-terminal GFP fusion proteins in N. salina. The ORF of PDK was amplified by PCR from the cDNA using the primers PDK_KpnI_f and PDK_KpnI_r. The PCR products were inserted into the KpnI sites, which reside immediately upstream of the eGFP sequence within the pPha-VCP-eGFP vector. The vector was introduced into wild-type N. salina by electroporation using Bio-Rad GenePulser Xcell apparatus (Bio-Rad, USA) at 12,000 V/cm, 50 µFD and 600 Ω. Zeocin (Invitrogen, USA) at the final concentration of 2 μg/ml (agar medium) and 1 μg/ml (liquid medium) was used for selecting transgenic cells. GFP positive cells were observed using a Leica TCS SP8 laser scanning confocal microscope.