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Basic Microbiology
Published in Philip A. Geis, Cosmetic Microbiology, 2020
The bacteria share several common structural characteristics with eukaryotic cells such as an outer cell membrane and an internal cytosol. In bacteria, together these are referred to as the “protoplast”. However, whereas eukaryotic cells have internal membrane-bound organelles (e.g., the nucleus), bacteria lack these internal organelles and hence the moniker “prokaryotes”, reflecting a common belief that eukaryotic cells evolved from such progenitors. Several observable bacterial cell structures as well as their composition and function(s) are listed in the section that follows (see Figure 1.3). Special attention is given to the bacterial cell wall.
Protoplasts as Tools in the Study of Moss Development
Published in R. N. Chopra, Satish C. Bhatla, Bryophyte Development: Physiology and Biochemistry, 2019
Neil W. Ashton, Philip J. Boyd, David J. Cove, Celia D. Knight
In this review we shall, for the sake of brevity, adopt the now common usage of the word “protoplast” to refer to the isolated protoplast; we shall not confine our use of the term “protoplast” to isolated protoplasts which have been examined critically for the complete removal of cell wall material. We will therefore be using “protoplast” where more critically we should use the term “isolated spheroplast”.
Biology of microbes
Published in Philip A. Geis, Cosmetic Microbiology, 2006
Cytoplasm, mesosomes, ribosomes, and other inclusions. A bacterial cell minus its wall is a protoplast. A protoplast includes the plasma membrane, the cytoplasm, and everything within it. The prokaryotic cytoplasm, however, does not have typical unit membrane-bound internal organelles. Within the cytoplasm is the nucleoid where the DNA genetic material is localized. Also, within the cytoplasm are the enzymes needed for growth and metabolism, the machinery for manufacturing those enzymes (ribosomes), and some internal membrane structures called mesosomes. Mesosomes are actually invaginations of the plasma membrane. Finally, some bacteria also contain inclusion bodies consisting of polyphosphate, cyanophycin, and glycogen. These inclusions are not usually membrane-bound. Other bacteria have inclusions bound by a single-layered nonunit membrane. These consist of poly-b-hydroxybutyrate, sulfur, carboxysomes, hydrocarbons, and gas vacuoles.
Successful production of human epidermal growth factor in tobacco chloroplasts in a biologically active conformation
Published in Growth Factors, 2023
Yunpeng Wang, Jieying Fan, Niaz Ahmad, Wen Xin, Zhengyi Wei, Shaochen Xing
For in planta observation, the transgenic plantlets and plants were exposed to 365 nm UV light in dark room and photographed. For laser confocal fluorescent microscope observation, the protoplasts were isolated in advance. The isolation of protoplasts was as followed: the leaves were cut into small slices and dipped into CPW buffer (27.2 mg L−1 of KH2PO4, 101.0 mg L−1 of KNO3, 1480.0 mg L−1 of CaCl2·2H2O, 246.0 mg L−1 of MgSO4·7H2O and 0.025 mg L−1 of CuSO4·5H2O, pH = 5.8) with 13% (m/v) mannitol for 30 min then the leave slices were sucked dry with filter paper and transferred to CPW buffer with 1% (m/v) cellulase Rs (Yakult Co., Japan) and 0.2% (m/v) pectinase Y23 (Yakult Co., Japan) and cultured at 28 °C, 60 rpm in the dark in incubator for 4 h. Protoplasts were harvested by centrifuge at 100 g, 4 °C for 2 min. The protoplasts were resuspended in CPW buffer and applied to laser fluorescent confocal microscope, and photos were shot by scanning the emissions excited by 554 nm and 488 nm laser every 0.2 μm in vertical dimension. The photo slice series were then merged.
Ethanol production from cassava starch by protoplast fusants of Wickerhamomyces anomalus and Galactomyces candidum
Published in Egyptian Journal of Basic and Applied Sciences, 2020
Tolulope Modupe Adeleye, Sharafadeen Olateju Kareem, Mobolaji O. Bankole, Olusegun Atanda, Abideen I. Adeogun
The protoplasts from the two isolates were mixed and suspended carefully in 35% polyethylene glycol (PEG) (Mol wt 3350), 10 mM CaCl2 and 0.8 M sorbitol. The suspension was incubated at room temperature (28ºC – 30ºC) for 30 min under the ultraviolet light. The resultant fused cells were washed with the protoplast buffer. One ml of the suspension was mixed with 10 ml of the regeneration medium (3% agar, 0.7% YPD and 0.8 M sorbitol). This was poured into plates containing a thin bottom layer of agar with the same regeneration medium composition. The plates were thereafter incubated at 30ºC for 3–7 days until visible regenerated colonies emerged. Parent and fusant colonies that emerged were purified and assayed for the desired recombination such as (i) flocculation, (ii) rate of fermentation in glucose and maltose broth cultures and (iii) ethanol tolerance. The preferred recombinants were thus selected and stored on sabouraud dextrose agar slants at 4°C. Parental and fusant yeasts were subcultured bi-monthly.
Enhanced production of tanshinone IIA in endophytic fungi Emericella foeniculicola by genome shuffling
Published in Pharmaceutical Biology, 2018
Pengyu Zhang, Yiting Lee, Xiying Wei, Jinlan Wu, Qingmei Liu, Shanning Wan
Genome shuffling was conducted with the aforementioned method (Zhang et al. 2002; Patnaik et al. 2002; Dai and Copley 2004; Hida et al. 2007) with some modifications. Samples of protoplasts (0.5 mL) were prepared by using hypertonic solution I (0.5 M KCl, 25 mM Tris–HCl buffer, pH 6.0) from two different strains which were mixed and equally divided into two aliquots. One aliquot was inactivated with UV light for 10 min, while the other one was heat treated at 50 °C for 50 min. Protoplasts inactivated by those two different methods were mixed, centrifuged, and resuspended in 1 mL of hypertonic solution II (0.5 M sucrose, 25 mM Tris–HCl buffer, pH 6.0). 9 mL of 30% PEG 4000 with 15% dimethyl sulfoxide (DMSO), and 10 mL of CaCl2 in buffer B was then added to the resuspended protoplast mixture and incubated for 7 min at 30 °C with the shaking speed of 100 rpm. The resultant fused protoplasts were centrifuged, washed for three times with hypertonic solution II, resuspended in 1 mL hypertonic solution II and then regenerated on regeneration medium plates in the incubator at 28 °C for 7 days. The fusion frequency was calculated as the ratio of the number of fusants from the protoplasts treated with PEG to the number of the regenerated strains without inactivation treatment. The large and high-yield tanshinone IIA-producing colonies were selected. Based on the screening and analysis methods described above, the best shuffled mutants were taken for the subsequent genome shuffling. The pooled fusion libraries were named as F1, F2 and F3, respectively.