This ligation resulted in a plasmid with mCitrine that proceeded with an in-frame HIV protease cleavage site (pHIVCLS-mCit). A cyan fluorescent protein, mCerulean (mCer), was PCR amplified from plasmid pEGFP-C1-mCerulean (Clontech Laboratories, Mountain View, CA, USA) using forward primer (5��-CATGGAATTCATGGTGAGCAAGGGCGAGGAGCTGTTCACC) and reverse primer (5��-CATGACTAGTCTTGTACAGCTCGTCCATGCCGAGAGTGATCC). The PCR product was cut using EcoRI and SpeI restriction enzymes and ligated into EcoRI and XbaI cut plasmid pHIVCLS-mCit to obtain an in-frame fusion of mCerulean-HIVCLS-mCitrine. The above-described process is a modified BioBrick cloning technique [13] that allows the creation of in-frame protein fusions.
The FRET-HIV sensor was cut from the BioBrick vector using restriction enzymes EcoRI and SpeI and ligated into the mammalian expression vector pcDNA3, which was cut with EcoRI and XbaI, thereby generating pFRET-HIV (Figure 1A).Figure 1.FRET-HIV protease-sensitive sensor. (a) The FRET-HIV sensor is composed of a donor mCerulean protein linked to an acceptor mCitrine with a peptide, which is a target site for the HIV protease. When excited with 433-nm light, the mCerulean emits light …A plasmid pCeVe (plasmid backbone pRSET-B) [14] coding for mCerulean linked to yellow fluorescent protein mVenus with an 8-amino acid linker (MHGGSGGTE) that was not cleaved by the HIV protease was used as the FRET-control plasmid. The FRET-control was used as a negative control for the in vitro HIV protease assays.
The pmCerulean and pmCitrine plasmids were used for donor- and acceptor-only controls, and for non-FRET control for the microscopy and flow cytometry. For in vivo expression of the HIV protease plasmid, pNL4-3.HSA.R?.E? (pNL4-3) was used [15,16].2.2. Cell Lines and TransfectionsHuman emb
In recent years, studies on human�Crobot interaction have needed to utilize tactile sensors to physically interact with people and their environment in contexts such as rehabilitation, home/hospital care, education, and entertainment. In regard to human communication, physical touch is essential for an infant or child’s social, cognitive, and physical development [1]. Touch also plays an important role in adulthood, when a person is soothing, playing, and maintaining proximity between a child and caretaker [2].
Carfilzomib Similarly, physical interaction with robots (such as hugging and hand shaking) builds closer relationships between a human and a robot from the perspective of spatial distance [3]. The ROBOSKIN project shows another application domain of tactile interaction, namely, that between robots and autistic children to improve social-interaction capabilities of the children [4]. Furthermore, physical therapy for stroke rehabilitation by robotic manipulators is a promising application of tactile sensors [5].
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