How are microorganisms cloned?

In the field of microbiology, cloning is quite common, such as simple cell division in single-celled organisms and multicellular organisms. The cloning technology of microorganisms is not complicated. Generally speaking, the growth of microorganisms needs a lot of water and more carbon sources to form an organic carbon framework and a nitrogen source for nitrogen-containing substances. Secondly, some salts containing phosphorus, magnesium, potassium, calcium, sodium, sulfur and trace elements such as iron, copper, zinc and manganese are needed. Different microorganisms have different requirements for nutrients. Some microorganisms are omnivorous and can use different substances as nutrition; Some microorganisms can use substances with relatively simple chemical composition, or even grow and develop in a completely inorganic environment, and synthesize their cellular substances by using inorganic salts such as carbon dioxide and ammonia. In addition, some microorganisms need some ready-made organic compounds such as vitamins, amino acids and purine bases to grow.

There are many types of microbial cell cultures. The so-called surface culture means using solid medium, and cells are located on the surface of solid medium. This culture method is mostly used for the separation, purification, preservation and seed preparation of strains. Microbiologists' laboratories often use surface culture, because although surface culture is simple in operation and equipment, it also has some shortcomings, such as it is difficult to keep the culture environment uniform.

Generally speaking, the method of surface culture is to dilute the suspension containing a variety of microorganisms to a certain proportion, then inoculate it on the solid slope of agar culture medium, and after incubation, isolated colonies can be obtained. The single colony can be formed by a single cell, thereby obtaining a pure cell line. The bacteria growing on the inclined plane can be preserved for 3 ~ 6 months at 4℃. When penicillin was first put into industrial production, this surface culture method was adopted.

If microbial cell culture is industrialized, surface culture is difficult to provide enough growth surface. As far as penicillin is concerned, if 1 kg penicillin is produced by surface culture, 1 culture bottle with a volume of 1 liter is needed. This requires a lot of manpower, energy and training space. Therefore, in industrial production, surface culture method was quickly replaced by deep culture method.

Submerged culture is a culture method suitable for mass production. Using the method of submerged culture, it is easy to obtain evenly mixed bacterial suspension, which is convenient for systematic monitoring and control. At the same time, the submerged culture method is also easy to scale up to industrial scale. Submerged culture method basically overcomes the shortcomings of surface culture method and becomes an important method to cultivate a large number of microorganisms. In submerged culture, bacteria are suspended in liquid culture medium, and oxygen in the air is introduced into cells through an aeration device.

Shake flask is often used in small batch deep culture in laboratory. Seal the bottle mouth with multi-layer gauze or polymer filter membrane to prevent miscellaneous bacteria or impurities in the air from entering the bottle. Air can enter the bottle through the bottle stopper for bacteria to breathe. The flask is filled with culture medium, sterilized, inoculated with bacteria, and then cultured on a shaker with heat preservation and vibration. Shake flask culture is a common method to obtain bacteria from experiments, and it is also used for seed culture in large-scale production.

In addition, to realize the automatic culture of industrial microbial cells, continuous culture is needed, because with the active growth of microorganisms, nutrients are continuously consumed and harmful metabolites are continuously accumulated, which can not maintain the logarithmic growth period for a long time. Therefore, in continuous culture, it is necessary to control the nutrient concentration and culture conditions to maintain the growth of microbial cells in logarithmic growth period. According to different control methods, continuous culture can be divided into constant turbidity method and constant method. In addition, the intermediate feeding batch culture method can also be implemented, that is, after the batch culture reaches a certain level, the culture medium is continuously or intermittently added to keep the concentration of restrictive matrix and cells in the culture basically unchanged.

In microbial cell culture, synchronous culture can not be ignored. In the synchronous culture method, by controlling the environmental conditions, the cell growth is at the same stage, so that all cells divide at the same time, that is, the cells in culture remain at the same growth stage. Synchronous culture is beneficial to understand the growth or physiological characteristics of a single microbial cell and the whole cell group.

In addition, through in-depth understanding of microbial growth and physiology, two or more microbial cells can be cultured in one culture tank at the same time. Under the condition of mixed culture, there are various relationships among microorganisms. One is irrelevant, and the growth of one microbial cell will not be changed because of the existence of another microbial cell, such as the constant culture of streptococcus and lactobacillus. The other is mutualism, in which two kinds of bacteria provide each other with nutrients needed for growth or consume their growth inhibitors. For example, Pseudomonas relies on methane as its only carbon source and energy source, and grows better when there are filamentous microorganisms. The methanol produced in the growth process of the former has adverse effects on its growth and respiration, while filamentous microorganisms can consume methanol and eliminate inhibition. Also, for example, bacteria can produce enzymes to break down antibiotics, so that their companions can grow. Compounds produced by other bacteria are carbon sources or energy sources for their peers, which is beneficial to their growth. This may be like "helping others" in human beings.