To evaluate the accumulation of PCB in hmox1 and ho1su1, the cyanobacterial bilin reporter NpF2164g5 from Nostoc punctiforme was fused with a twin-strep tag (STII) and inserted into the p322-PsbDp-AtpAp.aadA vector (Duanmu et al., 2013 (link)). The resulting plasmid was used for chloroplast transformation experiments with hmox1 and ho1su1 using particle bombardment with the PDS-1000/He system (BioRad, California, United States). The bombarded cells were transferred to a solid TAP medium containing 400 μg/mL spectinomycin. Positive transformants that expressed NpF2164g5-STII were identified using PCR and immunoblotting analyses and were further selected with at least 4∼5 rounds of growth on solid TAP medium supplemented with 400 μg/mL spectinomycin to achieve chloroplast DNA homoplasmy. Transgenic lines were named hmox1:NpF2164g5-STII and ho1su1:NpF2164g5-STII, respectively. NpF2164g5-STII purification was performed using Strep-tag II magnetic beads (Beaver, Suzhou, China) following the protocol recommended by the manufacturer. Purified NpF2164g5-STII protein was concentrated and used for zinc-dependent fluorescence assays to evaluate the presence or absence of covalently bound PCB as described previously (Zhang et al., 2018 (link)).
Pds 1000 he system
Manufactured by Bio-Rad
Sourced in United States, Canada
The PDS-1000/He system is a gene delivery device used for particle bombardment, also known as biolistic transformation. It utilizes high-speed helium gas to accelerate DNA-coated gold or tungsten particles into target cells or tissues. The system is designed for efficient and versatile gene delivery across a range of applications, including plant and animal cell transformation, gene expression studies, and genetic engineering.
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Lab products found in correlation
Bx51 epifluorescence microscope, by Olympus (5 mentions)
Dp71 digital camera, by Olympus (5 mentions)
Lsm 700, by Zeiss (4 mentions)
Dm6000b microscope, by Leica (2 mentions)
Fv1200, by Olympus (2 mentions)
Photo paint, by Corel (2 mentions)
Cell d software, by Olympus (2 mentions)
Tcs sp5 2, by Leica (2 mentions)
Pentr d topo vector, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Lsm 510 confocal microscopy system, by Zeiss (1 mentions)
Biolistic particle delivery system, by Bio-Rad (1 mentions)
Eclipse te300, by Nikon (1 mentions)
Lsm 410, by Zeiss (1 mentions)
Mrc 1024, by Bio-Rad (1 mentions)
Dm2500, by Leica (1 mentions)
Szx16, by Olympus (1 mentions)
Bx 60 of confocal laser scanning microscope, by Olympus (1 mentions)
Application suite advanced fluorescence lite, by Leica (1 mentions)
Sp8 x inverted confocal microscope, by Leica (1 mentions)
40 protocols using pds 1000 he system
1
Evaluating PCB Accumulation in hmox1 and ho1su1
2
Subcellular Localization of CrNPF2.9 and AtTPK1
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The full length CrNPF2.9 gene was subcloned into the pSCA-cassette vector as a YFP fusion, and the Arabidopsis AtTPK1 was subcloned into the into the pSCA-cassette vector as a CFP fusion. The vacuolar STR-CFP and the plasma membrane PM-CFP (CD3-1002) markers were described previously 5 (link),16 (link). All vectors used a 35S promoter for expression. Transient transformation of C. roseus cells by particle bombardment and fluorescence imaging were performed following the procedures previously described 5 (link),49 (link). C. roseus cells were bombarded with DNA-coated gold particles (1 μm) and 1,100 psi rupture disc at a stopping-screen-to-target distance of 6 cm, using the Bio-Rad PDS1000/He system and 100 ng of each plasmid per transformation. For the transient transformation of onion cells, internal epidermis of fresh onion purchased from local producer (Les Vergers de la Breteche, St-Paterne Racan), were peeled and placed on solid vitamin-free MS medium and bombarded following the same protocol. Both cell-types were cultivated for 16 h to 38 h before being harvested and observed. The subcellular localization was determined using an Olympus BX-51 epifluorescence microscope equipped with an Olympus DP-71 digital camera and a combination of YFP and CFP filters.
3
Subcellular Localization of OsWAK112
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To investigate the subcellular localization of OsWAK112, the coding sequence of OsWAK112 from OsWAK112/pENTR was introduced into binary vector pMDC83 to construct the vector 35S:: OsWAK112-GFP, and 35S::GFP was used as a negative control vector. Both constructs were transferred to A. tumefaciens strain GV3101 and then transiently transformed into tobacco (N. benthamiana) leaves or transformed into onion (Allium cepa) epidermal cells by particle bombardment using the Bio-Rad PDS-1000/He system according to the manufacturer’s protocol. After 40 to 48h infiltration, localization of the protein was examined using a Zeiss LSM 510 confocal microscopy system with a 488nm laser for excitation from 500 to 515nm for GFP emission. 0.9M mannitol was used to induce plasmolysis.
4
BpMYB106-GFP Fusion Construct Expression
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The whole coding sequence of BpMYB106, with removed stop code, was ligated with SalI and SpeI-digested pBI121 vector to generate pBI121-BpMYB106-GFP containing BpMYB106-GFP fusion construct under the control of CaMV 35S promoter. The construct was confirmed by sequencing and used for transient transformation of onion (Allium cepa) epidermis via a PDS-1000/He System (Bio-Rad, Hercules, CA, USA). After 24 h of incubation, GFP fluorescence in transformed onion cells was observed under a confocal laser microscope (Zeiss LSM700, Jena, Germany).
5
Subcellular Localization of SmbHLH51
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The subcellular localization assay was performed by transiently expressing the SmbHLH51-GFP fusion protein in onion epidermal cells. The entire CDS region of SmbHLH51 was amplified from pMD19-cSmbHLH51 with primers 207-SmbHLH51-F/R, which contain the attB1/attB2 sites. The PCR products were cloned into entry vector pDONR207, using the BP recombination reaction, and then transferred into destination vector pEarleyGate103 (Earley et al., 2006 (link)) through an LR reaction, according to the protocol from the Gateway technology manufacturer (Invitrogen, United States). The pEarleyGate103-SmbHLH51 vector was transformed into onion epidermal cells by particle bombardment at a helium pressure of 1100 psi with the PDS-1000/He system (Bio-Rad, United States) and observed using a Leica DM6000B microscope (Leica, Germany) as described before (Li et al., 2018 (link)). The pEarleyGate103 plasmid was transformed into onion epidermal cells as a positive control.
6
Transient Expression of MiMsp40 in Onion Cells
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The MiMsp40 gene with and without signal peptide-encoding regions was amplified using the gene-specific primer pairs M40-F1 KpnI/M40- R1 XbaI and M40-F2 KpnI/M40- R1 XbaI, containing KpnI and XbaI restriction enzyme sites in the forward and reverse primers, respectively (Supplemental Table S2 ). The resulting amplified fragments were cloned into the respective sites in a modified p35SeGFP vector between the cauliflower mosaic virus (CaMV) 35S promoter and enhanced green fluorescent protein (eGFP) to express the eGFP fusion protein; the p35SeGFP vector without MiMsp40 was used as a control. Both constructs were confirmed by DNA sequencing. The resulting constructs were introduced into onion (Allium cepa) epidermal cells by biolistic bombardment with a PDS1000/He system (Biolistic Particle Delivery System, Bio-Rad, CA, USA). Onion pieces were incubated for 24 h at 24 °C in the dark; epidermal peels were then observed through a fluorescence microscope (Nikon Eclipse TE300, Tokyo, Japan).
7
Constructing CaMV 35S::PsnCYCD1;1-GFP Fusion Gene
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The full-length PsnCYCD1;1 coding region (without stop codon) was amplified from pMD18-T-PsnCYCD1;1 by PCR, digested with Sal I and Nco I, collected the target fragments, and directionally ligated into vector pUC18 to construct the CaMV 35S::PsnCYCD1;1-GFP fusion gene. The primers used to amplify PsnCYCD1;1 are listed in Supplementary Table S1 . The PsnCYCD1;1-GFP recombinants and empty vectors were transiently transformed into onion epidermal cells by the gene gun method (Bio-Rad PDS-1000/He System, USA). The green fluorescent signal of the PsnCYCD1;1-GFP fusion proteins was observed and photographed using laser confocal microscopy (model LSM410, Zeiss, Jena, Germany).
8
Onion Epidermal Cell Transformation
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The fusion pCaMV35S::TaADF3-GFP construct and the control plasmid pCaMV35S::GFP were transformed into onion epidermal cells by particle bombardment at a helium pressure of 1100 psi using the PDS-1000/He system (Bio-Rad, Hercules, CA, USA). The transformed onion epidermal cells were cultured on MS medium plates at 28°C for 18–24 h in a dark chamber. Fluorescent signals were observed using a Zeiss LSM 510 confocal laser microscope (Zeiss, Germany) with a 480-nm filter.
9
Visualizing Dlf1 Localization in Onion Cells
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The coding sequence of Dlf1 was amplified and fused in frame to the upstream of green fluorescent protein (GFP) gene to generate the CamUbi:Dlf1-GFP construct. The resultant and the control CamUbi:GFP vectors were transformed into onion (Allium cepa) inner epidermal cells by bombardment using the PDS-1000/He system (Bio-Rad) with DNA-coated gold particles. The transformed cells were cultured on 1/2 MS medium at 28°C for 2 d and observed under a confocal microscope (Bio-Rad MRC 1024).
10
Optimization of DNA Delivery Parameters
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To optimize DNA delivery parameters of the Bio-Rad PDS-1000/He System, two sizes of gold particles (0.6 or 1 µm), two Helium pressures (900 or 1100 psi), and two target distances from the stopping mesh to the calli (9 or 12 cm) were applied in this experiment. A total of eight combinations of different parameters were assessed (Table 1 ). As for each combination, 27 calli were tested in triplicate. The GUS staining was performed by placing the transformed calli, and the control calli, into GUS staining solution three days post-bombardment, with the calli incubated in the staining solution for 24 h (h) at 37 °C. Post this incubation period, the number of callus with blue foci was recorded.
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