As a novice experimenter, many times the concentration of plasmids is very different, some are as high as 500~600ng/uL, and some are only a few dozen. All the same strains and kits are used. Why is there such a big difference in the concentration of small extracts?
To solve this problem, we must first understand the plasmid. '
Copy number
For plasmid vectors, copy number is one of the characteristics we are most concerned about. In fact, the copy number of the plasmid in each bacterium is mainly determined by the replication characteristics of the plasmid itself. According to the nature of replication, plasmids can be divided into two categories:
Stringent plasmid : When the bacterial chromosome replicates once, the plasmid also replicates once, and each bacterium contains only 1 to 2 plasmids;
Relaxed plasmid : When the bacterial chromosome replication stops, it can continue to replicate. Each bacteria generally contains about 20 plasmid copies. The replication of these plasmids is under the loose control of the host. Each bacterium contains 10-200 copies. If a certain drug is used to inhibit the synthesis of host protein, the number of plasmid copies can be increased to several thousand copies.
Of course, the constant copy number is related to the plasmid replication control system, the size of the plasmid, and the culture conditions.
So here you may ask, we can mention more plasmids for high-copy plasmids. What is the role of low-copy-number plasmids? Indeed, low-copy number plasmids are not very versatile, and are mainly used for the following two points:
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Plasmids with high copy numbers are often unstable, and low copies are used when cloning large fragments or cloning with toxic DNA;
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The amplification of plasmids will take up a lot of resources. When vectors are used for expression or other purposes, low-copy plasmids are also used.
Conjugative transfer and shuttle plasmid
Conjugative transfer of plasmids : It is an important method for the transfer of bacterial genetic material. In the process of plasmid transfer, the donor bacteria and the recipient bacteria are in close contact by binding, the plasmid is transferred from the donor cell to the recipient, and the plasmid is replicated at the same time. According to whether they can be transferred autonomously, naturally-occurring plasmids can be divided into two categories: transfer-type plasmids and non-transfer-type plasmids.
It should be noted here that the bacteria that obtain the plasmid can subsequently acquire some biological characteristics, such as drug resistance or the ability to produce bacteriocins. From the perspective of environmental friendliness, the waste bacteria liquid in the laboratory must be sterilized before it can be poured.
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Source: 51xxziyuan.com