Btx ecm 830 system

A sample for in-cell NMR measurement was prepared as described in (16 (link)). Briefly, HeLa cells were electroporated with 400 μM non-labelled DNA oligonucleotide (d-AAGGGTGGGTCCCACCCTGGGTGGGT) supplemented with 10 μM 5′-FAM labelled oligonucleotide in electroporation buffer (140 mM Na₂HPO₄/NaH₂PO₄, 5 mM KCl, 10 mM MgCl₂, pH 7.0), using electric pulses 1000 V, 100 μs followed by 350 V, 30 ms (BTX-ECM 830 system; Harvard Apparatus, USA). After electroporation, FAM intensity in cells was monitored with flow cytometry (BD FACS Verse Cell Analyzer; BD Biosciences, USA) to check transfection efficiency and propidium iodide staining was used to determine cell survival. Subcellular localization of the introduced oligonucleotide and cell morphology was monitored using confocal microscopy (LSM 800; Carl Zeiss, Germany). For in-cell NMR, the cells were suspended in Leibovitz L15–/– medium containing 10% D₂O, transferred into a 5 mm NMR cuvette, and pelleted by manual centrifugation to concentrate in active coil volume. 1D ¹H in-cell NMR spectra were acquired at Bruker Avance NEO 950 MHz NMR (Bruker Corporation, Billerica, MA, USA) using a 1D ¹H JR-echo (1–1 echo) pulse sequence (17 ) with zero excitation set to the resonance of water and the excitation maximum set to 12.5 ppm.

We prepared several mutants of strain No. 10, as shown in Table 1. Notably, allelic replacement and gene complementation were performed as previously described (65 (link)). The plasmids and primers used in this study are listed in Table 2; Table S1. Furthermore, vector DNA for transformation was extracted from RN4220 using the manual alkaline SDS method or from BL21 using the FastGene Plasmid Mini Kit (Nippon Genetics Co., Ltd., Tokyo, Japan). Thereafter, electroporation was performed using the ELEPO21 system (NEPA GENE, Chiba, Japan) or BTX ECM830 system (BTX Harvard Apparatus, Inc., Holliston, MA, USA).

For overexpression of mNeon-Ty tagged proteins, we utilized (i) the original pTREX vector (28 (link)) modified with the T. cruzi-optimized and “fixed” neomycin resistance cassette as previously described (26 (link)); (ii) the minimal pTREX vector that we had generated, pMiniTREX (see Fig. S4A in the supplemental material); or (iii) pTMiniTREX with the BBa_B1006 terminator (58 ) upstream of the insertion site (Fig. S4C). Final vectors were assembled using an NEB HiFi assembly kit. Parasites were transfected as described previously by Lander et al. (59 (link)) using a BTX ECM 830 system (Harvard Apparatus). However, only two electroporation pulses were used for transient transfections to reduce parasite loss for 48 h posttransfection imaging. A list of the DNA primers used during this work can be found in Table S1 in the supplemental material.