Plasmids are circular DNA molecules that are present inside the bacteria, containing its genetic material, independent of the chromosomal DNA. Moreover, plasmids are capable of replicating on an individual basis.
Two Properties of Plasmids
It has been found that plasmids with linear structures are composed of RNA. Plasmids are found either as a single copy, however, multiple copies of plasmids can be found in one bacterium. Similarly, the number of genes present inside can vary from six to more than a hundred sets.
Plasmids are found in bacteria in great numbers, as matter of fact, there is barely any plasmid that lives outside the bacteria, therefore it is not shocking to reveal that more than 50% of bacteria found in nature had bacteria with one or multiple plasmids. Bacteria are not the only organism inhabited by plasmids, the bigger organisms like Fungi and yeast sometimes are also hosts to these entities.
The number of copies of the plasmid in each bacteria is known as the copy number. Most of the plasmids have one to two copies of plasmid, however, the copy number for some plasmids like CoIE can be as many as 50. What copy numbers represent is the strength of plasmid-borne characteristics, such as antibiotic resistance. The greater the copy number is in a cell, the higher would be antibiotic resistance genes and consequently, there will be greater resistance of antibiotics in those bacteria.
If we talk about the size of plasmids, then it greatly varies. For instance, the F-plasmid of E.coli is 1% of the total size of the chromosome present in E.coli. However, most of the plasmids found are much smaller in size. Though there are plasmids that are about 10% the size of their neighboring chromosome, those big plasmids are a rarity.
Why Are Plasmids Beneficial to Bacteria?
As mentioned earlier, plasmids carry a range of genes that allows them to manage and carry their own life cycle. Furthermore, they create resistance against the host cell, if they try to attack the bacteria. There are certain plasmids known as cryptic plasmids, which leave no trace for their identification. Cryptic plasmids, as the name suggests, carry genes that are still unknown. Microbiologists use plasmids to carry research in genetic engineering.
The host of the plasmids also are very diverse. There are some plasmids such as F.plasmid of E.coli, which are inhabited by some other bacteria as well, such as the Salmonella and Shigella. There are also some plasmids that have nearly a hundred bacteria hosting them, for instance, plasmids of the P-family can live in many various species of bacteria. The “P” was named after “Pseudomonas”, however, it is now being regarded as “promiscuous”. These are usually helpful for creating a wide range of antibiotic resistance.
There are many medium-sized bacteria that can move from one bacterial cell to another, this is known as transferability. Plasmids of F-type and P-type can very easily transfer, therefore they are also called transfer-positive. However, the transferability requires at least 30 genes, making very few numbers of plasmids that can transfer (Clark, 2019).
Increasing the yield of plasmids is not too hard, however, a keen eye and informatics mind is crucial to speed up the replication process. Although, right after the excitement of growing a plasmid and isolating its DNA, fades away, the rest of the process becomes very tedious. This when it gets tough to extract the maximum yield of plasmid DNA out of the culture you have made.
1. Try Chloramphenicol There are a number of vectors with very relaxed replication origin, this permits the decoupling protein synthesis from the replication of plasmid present in E.coli. If you add chloramphenicol, it will halt the protein synthesis, yet plasmid inside will continue to replicate. This will give you a large number of copies of your vector in every bacterial genome. There are more relaxed origins of plasmids and there are a couple of ways to use chloramphenicol for the amplification of the plasmids. a. Use it according to “The Maniatis” This method suggests letting the culture grow until the saturation level is achieved. Add (after measuring 170 micrograms) chloramphenicol and allow the culture to grow further for a duration of 16 hours. This will ensure the protein synthesis is stopped, but the number of the vector keeps increasing. b. Use it according to Begbie This method ensures the faster possibility of gaining the maximum yield. The previous procedure took 36 hours in total. Add the concentration of chloramphenicol as low as 3 micrograms per millilitre. The partial or sub inhibition will occur, slowing the time and doubling the copy number of E.Coli, rather than completely stopping the replication. The copy number of your vector will also rise. Whichever method suits you, to boost the number of vectors by using the chloramphenicol, you should treat the product well, as it contains high copies of vectors. Don’t overload maxiprep or midiprep with lysate and try using a minimum volume of the culture described by the protocol. Repeat the elution after getting the maximum value of the buffer. Feel free to keep on concentrating the DNA later. 2. Increase the pressure to gain more Plasmids Don’t forget that you are always needed to maintain the optimal pressure in the culture of your plasmid. Or else, you will find that plasmid is reducing from the culture. Therefore, always remember to add antibiotics or just any appropriate agent in the medium, while growing the culture. In case you are choosing to add ampicillin as a selective agent, you need to know that the plasmid will produce enzymes that trigger ampicillin-destruction. Moreover, it also gives beta-lactamase, which will accumulate inside the medium during the growth phase. In order to remove the beta-lactamase, you need to get rid of the culture medium via re-suspending them in the freshly made medium or pelting them before starting to scale the culture (Doronina, 2016). 3. Try to improve the culture media Adding supplements to get a richer medium is the easiest and the most efficient way of improving cell density. As culture content plays an integral part in the yield production of DNA. However, different strains and plasmid will vary in growth (Hazen, 2016).
Clark, D. P. (2019). Plasmids. Elsevier Inc.
Doronina, V. (2016, August 23 ). How to Improve Plasmid Yield Using Antibiotics. Retrieved from BiteSizeBio: https://bitesizebio.com/30049/improving-plasmid-yield-antibiotics/
Hazen, A. (2016, May 26). Plasmids 101: Optimizing Plasmid Yields. Retrieved from add gene blog: https://blog.addgene.org/optimizing-plasmids-101-plasmid-yields