Spheroplast
Encyclopedia
A spheroplast is a cell from which the cell wall
has been almost completely removed, as by the action of penicillin
. The name stems from the fact that after a microbe's cell wall is digested, membrane tension causes the cell to acquire a characteristic spherical shape. Spheroplasts are osmotically fragile, and will lyse
if transferred to a hypotonic solution.
bacteria
can be used to study the function of bacterial ion channels through a technique called patch clamp
, which was originally designed for characterizing the behavior of neurons and other excitable cells. To prepare giant spheroplasts, bacteria are grown in a medium containing chemicals that prevent the cells from dividing completely. This causes bacteria to form long "snakes" that share a single membrane and cytoplasm
. After a period of time, the cell walls of the "snakes" are digested, and the bacteria collapse into very large spheres surrounded by a single lipid bilayer. The membrane can then be analyzed on a patch clamp
apparatus to determine the phenotype
of the ion channels embedded in it. It is also common to overexpress a particular channel to amplify its effect and make it easier to characterize.
The technique of patch clamping giant E. coli spheroplast have been used extensively for studying the native mechanosensitive channels (MscL, MscS, and MscM) of E. coli since 1987. Recently, it has been extended to study other heterologously expressed ion channels and has been shown that the giant E. coli spheroplast can be used as an ion-channel expression system comparable to Xenopus
oocyte
.
cells are normally protected by a thick cell wall
which makes extraction of cellular proteins difficult. Enzymatic digestion of the cell wall with zymolyase, creating spheroplasts, renders the cells vulnerable to easy lysis
with detergents or rapid osmolar pressure changes.
animal cells. Spheroplasts with recombinant DNA are introduced into the media containing animal cells and are fused by polyethylene glycol
(PEG). With this methodology, nearly 100% of the animal cells may take up the foreign DNA.
Cell wall
The cell wall is the tough, usually flexible but sometimes fairly rigid layer that surrounds some types of cells. It is located outside the cell membrane and provides these cells with structural support and protection, and also acts as a filtering mechanism. A major function of the cell wall is to...
has been almost completely removed, as by the action of penicillin
Penicillin
Penicillin is a group of antibiotics derived from Penicillium fungi. They include penicillin G, procaine penicillin, benzathine penicillin, and penicillin V....
. The name stems from the fact that after a microbe's cell wall is digested, membrane tension causes the cell to acquire a characteristic spherical shape. Spheroplasts are osmotically fragile, and will lyse
Lysis
Lysis refers to the breaking down of a cell, often by viral, enzymic, or osmotic mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a "lysate"....
if transferred to a hypotonic solution.
Patch clamping
Specially prepared giant spheroplasts of Gram-negativeGram-negative
Gram-negative bacteria are bacteria that do not retain crystal violet dye in the Gram staining protocol. In a Gram stain test, a counterstain is added after the crystal violet, coloring all Gram-negative bacteria with a red or pink color...
bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
can be used to study the function of bacterial ion channels through a technique called patch clamp
Patch clamp
The patch clamp technique is a laboratory technique in electrophysiology that allows the study of single or multiple ion channels in cells. The technique can be applied to a wide variety of cells, but is especially useful in the study of excitable cells such as neurons, cardiomyocytes, muscle...
, which was originally designed for characterizing the behavior of neurons and other excitable cells. To prepare giant spheroplasts, bacteria are grown in a medium containing chemicals that prevent the cells from dividing completely. This causes bacteria to form long "snakes" that share a single membrane and cytoplasm
Cytoplasm
The cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...
. After a period of time, the cell walls of the "snakes" are digested, and the bacteria collapse into very large spheres surrounded by a single lipid bilayer. The membrane can then be analyzed on a patch clamp
Patch clamp
The patch clamp technique is a laboratory technique in electrophysiology that allows the study of single or multiple ion channels in cells. The technique can be applied to a wide variety of cells, but is especially useful in the study of excitable cells such as neurons, cardiomyocytes, muscle...
apparatus to determine the phenotype
Phenotype
A phenotype is an organism's observable characteristics or traits: such as its morphology, development, biochemical or physiological properties, behavior, and products of behavior...
of the ion channels embedded in it. It is also common to overexpress a particular channel to amplify its effect and make it easier to characterize.
The technique of patch clamping giant E. coli spheroplast have been used extensively for studying the native mechanosensitive channels (MscL, MscS, and MscM) of E. coli since 1987. Recently, it has been extended to study other heterologously expressed ion channels and has been shown that the giant E. coli spheroplast can be used as an ion-channel expression system comparable to Xenopus
Xenopus
Xenopus is a genus of highly aquatic frogs native to Sub-Saharan Africa. There are 19 species in the Xenopus genus...
oocyte
Oocyte
An oocyte, ovocyte, or rarely ocyte, is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is produced in the ovary during female gametogenesis. The female germ cells produce a primordial germ cell which undergoes a mitotic...
.
Cell lysis
YeastYeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...
cells are normally protected by a thick cell wall
Cell wall
The cell wall is the tough, usually flexible but sometimes fairly rigid layer that surrounds some types of cells. It is located outside the cell membrane and provides these cells with structural support and protection, and also acts as a filtering mechanism. A major function of the cell wall is to...
which makes extraction of cellular proteins difficult. Enzymatic digestion of the cell wall with zymolyase, creating spheroplasts, renders the cells vulnerable to easy lysis
Lysis
Lysis refers to the breaking down of a cell, often by viral, enzymic, or osmotic mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a "lysate"....
with detergents or rapid osmolar pressure changes.
Transfection
Bacterial spheroplasts, with suitable recombinant DNA inserted into it, can be used to transfectTransfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
animal cells. Spheroplasts with recombinant DNA are introduced into the media containing animal cells and are fused by polyethylene glycol
Polyethylene glycol
Polyethylene glycol is a polyether compound with many applications from industrial manufacturing to medicine. It has also been known as polyethylene oxide or polyoxyethylene , depending on its molecular weight, and under the tradename Carbowax.-Available forms:PEG, PEO, or POE refers to an...
(PEG). With this methodology, nearly 100% of the animal cells may take up the foreign DNA.