Why do plasmids replicate on their own?

Why do plasmids replicate on their own?

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I know how plasmids can replicate independently of the main genome and know that they confer various properties to the bacteria and are useful in conjugation.

My question is - what is the advantage of plasmid replicating on their own? (Would there be any disadvantage if they were controlled by the main genome instead?)

In plasmids with high number of copies, the plasmid needs a certain density to be able to supply its function. If a given cell contains a few dozens of plasmid mollecules, and this is its optimal density, the easiest way to achieve this is to downregulate the replication by its own presence or the presence of one of its components. This also allows a random segregation during cell division, due basically by simple diffusion. It's also easier for the plasmid to achieve its optimal density once it has enter in a new cell by conjugation or transformation.

Plasmids are also subject to selection, so it is reasonable to assume that the more independent and more contagious a plasmid is, the bigger its presence in the population. In fact, it is a reasonable hypothesis to assume that many viruses are parasitic plasmids (many viruses in fact have a similar genomic structure and share many traits with them).

However, in plasmids with low number of copies the replication events can be dependent of the genome replication as you describe (despite the fact that many plasmids of this kind still regulate its own replication by itself). In this case, though, the line between plasmid and chromosome is less clear. In some organisms some big plasmids seems to form part of the genomic organization in the whole strain, and they usually contain important genes. This plasmids behave just like regular chromosomes, and they segregate by the same mechanisms the rest of the genome use. For those plasmids the terms minichromosome and megaplasmid are used, and the choice usually depends of the kind of genes it contains.

It is also an interesting thought that plasmids may be pieces of DNA that light be "parasites" on the bacterial cell which they use to provide resources for replicating themselves.

Plasmid copy number

In cellular biology, the plasmid copy number is the number of copies of a given plasmid in a cell. To ensure survival and thus the continued propagation of the plasmid, they must regulate their copy number. If a plasmid has too high of a copy number, they may excessively burden their host by occupying too much cellular machinery and using too much energy. On the other hand, too low of a copy number may result in the plasmid not being present in all of their host's progeny. Plasmids may be either high copy number plasmids or low copy number plasmids the regulation mechanisms between these two types are often significantly different. Biotechnology applications may involve engineering plasmids to allow a very high copy number. For example, pBR322 is a low copy number plasmid (

20 copies/cell) from which several very high copy number cloning vectors (

1000 copies/cell) have been derived. [1]

Types of origins of replication

There are lots of origins of replication out there so, for simplicity’s sake, we've ignored those used in eukaryotic cells and viruses and focused only on those found in bacteria. Some common ones you might see include ColE1, pMB1 (which comes in a few slightly different but well known derivatives), pSC101, R6K, and 15A. Not all origins of replication are created equal. Some will produce many plasmid copies and others produce just a few copies depending on how they are regulated. Generally, control of replication is referred to as "relaxed" or "stringent" depending on whether the ori is positively regulated by RNA or proteins, respectively. A plasmid's copy number has to do with the balance between positive and negative regulation and can be manipulated with mutations in the replicon. For example, the pMB1 ori maintains about 20 copies per cell, while pUC – which differs by only two mutations – will produce as many as 700 copies per cell.

Figure 1: A plasmid map showing the standard features of a plasmid.

So, how do you choose? Addgene Senior Scientist Marcy Patrick says researchers can ask themselves a few questions to get started:

  • Will the plasmid be used exclusively in E. coli? Gram negative bacteria in general? Both Gram negatives and Gram positives?
  • Will you have only one plasmid type in your cells at a time?
  • Do you want to make a lot of your plasmid?
  • Is the gene toxic in high amounts? It is always good to k eep in mind that plasmids with low to medium copy numbers can still express massive amounts of protein given the proper promoter and growth conditions.

Plasmid - Defination, Structure, Properties, replication, Copy number, Types, Functions and applications

Plasmid is a short, naturally occurring extra chromosomal, usually circular, double stranded DNA molecule that replicate, autonomously and lead an independent existence in Bacterial cell.

The term Plasmid was first given by Joshua Lederberg in 1952.

Properties of Plasmid :

• Plasmid is naturally found in cytoplasm, seperately from the main bacterial chromosome and are much smaller incomparision.

• Plasmids are mostly circular negatively supercoiled, double stranded DNA molecule. But linear Plasmids are also reported in genera streptomycetes and Borrelia. Also RNA plasmids are rare but reported in few plants, fungi and animal.

• Plasmids are normally in size range of 1kb to 250 kb which is smaller much smaller than Bacterial chromosome.
• Plasmids are considered as replicon. They replicate independently and code for their own transfer (i.e. Ori site present).
Smaller plasmid use host cell DNA replication machinary.
Larger plasmid carry gene for special enzymes specific for plasmid replication.

• Some are interogative plasmid called episomes. Also able to replicate by inserting them into bacterial chromosome and may be stably maintained in this form through numerous cell division, but show independent existence at some stage.

Copy number : Number of plasmids in an individual bacterial cell may very (1-100 or more) denoted by copy number.

• Plasmids are not essential for viability of bacterial cell. but genes carried by plasmids usually encodes traits beneficial for bacteria. e.g. antibiotic resistance, heavy metal resistance, Virulence factors, N2 fixation.

• Plasmid provide a mechanism of horizontal gene transfer via conjugation, transduction and transformation
Examples of plasmids : Puc 8 (E.cli), R-1, R-6, Col E1 (E.coli), Tol (Pseudomonas putida).

General Structure of Plasmid :

Structurel elements of bacterial plasmids may vary according to their size and function. Plasmid have following elements -

Structure of Plasmid

1. Origin of replication : DNA sequence which encode initiation of plasmid replication by recruiting bacterial transcription machinary for replication enzymes & proteins.

2. Antibiotic resistance gene : these genes provide a survival advantage to the bacterial host thet allows for selection of plasmid containing bacteria.

3. Multiple cloning site : Short segment containing a several restriction enzyme sites enabling easy insertion of foreign DNA.

4. Promoter region : It drives the transcription of the foreign insert.

5. Selectable marker : It is used to select for cells that has successfully taken up plasmid for the purpose of expression of inserted DNA.

6. Primer binding site : It is used as an initiation point for PCR amplification or sequencing of the plasmid.

Types of Plasmid (based on function) -

A. Fertility or F-plasmid :

B. Resistance or R-plasmid:

C. Col plasmid :

D. Metabolic/Degradative Plasmid :

E. Virulence Plasmid :

F. Suicide plasmid :

Types of plasmid based on their ability to transfer to another bacteria -

B. Non-conjugative plasmid -
This plasmid incapable of initiating conjugation, hence can only be transferred with the help of conjugative plasmid (tra-, mob-) under some circumstances.
E.g. many R plasmid, most Col plasmid.

C. Mobilisable plasmid :
This is an intermediate class of plasmid carry only a subset of genes (mob+) required for transfer.
They parasitize another plasmid, transferring at high frequency in presence of conjugative plasmid
(tra+, mob+).

Plasmid replication :

Plasmid are replicate autonomously and it also contain origin of replication site. There are two models are proposed for plasmid replication -
1) Theta θ model :
2) Rolling circle model :

1). Theta θ model :

Theta model replication of plasmid

2). Rolling circle model :

Host range :

Copy Number :

Copy number refers to the number of plasmid molecules of an individual.
Plasmid that are normally found in a single bacterial cell.
Plasmids are Classifying into two types based on copy no.
1). Stringent Plasmid :
- It is a large plasmid molecule.
- have low copy no. It present 1 to 2 per cell.
2). Relaxed plasmid :
- This plasmids are very small.
- have high copy no. 50 or more per cell.

Regulation of copy number :

Copy number is regulated by negative regulatory mechanisms and adjusting rate of plasmid replication.

1, by antisense RNA counter transcribes RNA,- plasmid replication is controlled by regulating the amount of available primer for replication.

2, Regulation by binding of replication proteins to introns (18-22 bp repeated units). Regulating amount of replication machinary available.

3, Regulation by ct RNA and protein, counter transcribed RNA limits the function of essential replication protein.

Plasmid incompatibility :

Incompatibility is defined as inability of two closely related plasmids to co-exist stably in the same host cell.
Several different kinds of plasmids may be found in a single cell, including more than one different conjugative plasmid.
To be able to coexist in the same cell, different plasmids must be compatible.
Different types of plasmids can therefore be assigned to different incompatibility groups, on the basis of whether or not they can coexist.

Incompatibility group : two plasmids that can not coexist in the same bacterial cell belong to a incompatibility group.

Why plasmids are incompatible ?

Partitioning :

It is active process that ensure that after cell division each daughter cell gets at least one copy of plasmid.
- Relaxed plasmid each daughter cell after division may get a copy by random diffusion or segregation.
- Stringent Plasmid most likely one daughter cell not received plasmid during segregation.

Par ABS system :- it is a broadly conserved molecular mechanisms for plasmid partitioning and chromosome segregation in bacteria.

Par ABS has 3 elements -
Par A - ATPas
Par B - DNA binding protein
Both are found on same operon.
Par S - cis acting sequence, located within or adjacent to this operon.

Confirmation of Plasmids :

Functions/Uses of plasmid (in genetic engineering) :

Advantages of using plasmid in genetic engineering :

Plasmid in eukaryotes :

- circular plasmid which is 6.3 kbcircular found in most Saccharomyces cerevisiae strain.
- 50-100 copies per haploid genome of yeast.
- This is the mostly studied eukaryotic plasmid.


Staphylococcus aureus is an opportunistic pathogen that causes both superficial and invasive infections, such as sepsis, endocarditis, pneumonia and osteomyelitis. Due to the frequent emergence of strains with plasmid-encoded antibiotic-resistance, it is important to understand how S. aureus plasmids are transferred and maintained.

The plasmid pSA564a belongs to the large &beta-lactamase/heavy-metal resistance plasmid family, which can also be found in feared MRSA strains such as S. aureus N315 and the community-acquired S. aureus USA300. Many plasmids from this family provide resistance to multiple antibiotics, with pSK1 encoding trimethoprim, quaternary ammonium, gentamicin, tobramycin and kanamycin resistance genes. Fortunately, these plasmids all appear to have a very narrow host-range, which limits their occurrence to S. aureus, and suggest the existence of a host-factor that is required for their replication.

A small plasmid-encoded transcript (RNA1) was identified anti-sense to the 5'-UTR of the RepA replication initiation gene in pSK1 and pN315, and this RNA1 was shown to inhibit translation of the RepA protein in pSK1. Our lab recently discovered that deletion of the 5' to 3' exoribonuclease J1 (RNase J1) results in the loss of the otherwise stably maintained pSA564a. By using a vector that replicates independently of the pSA564a origin of replication, we can show that the RNA1 accumulates in the RNase J1 mutant, suggesting that efficient degradation of the RNA1 is essential for replication of pSA564a. This hypothesis was further strengthened by cloning the region encoding RNA1 on a multi-copy plasmid, which led to rapid elimination of pSA564a. RNase J1 is a major player in RNA decay of Gram-positive bacteria, but has no ortholog in E. coli, perhaps explaining why the ßlactamase/heavy-metal resistance plasmids have such a narrow host range.

The DEAD-box helicase CshA and the 3' to 5' exonuclease PNPase are also factors in RNA decay, and it was therefore surprising that a RNase J1, CshA, PNPase triple mutant, although extremely sick, nevertheless maintains pSA564a replication.

Full-genome sequencing was used to verify that pSA564a was not integrated into the chromosome of the triple mutant, but is indeed kept as an episome, which should be reliant on its own origin of replication. Complementing the triple mutant with wild-type CshA led to immediate loss of pSA564a, whereas a CshAK52A active-site mutant did not, showing that the helicase activity of CshA is an important factor for plasmid replication.

Current work is focused on confirming that RNase J1 is needed as host-factor for other members of the pSA564a plasmid family, and to determine the RNase J1 mediated decay-path of the small regulatory RNA1. Furthermore, we are examining the molecular mechanism by which the deletion of CshA counteracts the effects of the RNase J1 deletion, and whether CshA directly modifies the formation of secondary structures within the RNA1, the 5'-UTR target, or the interaction between the two.

Old is New?: New horizon of conjugation in the generation of synthetic biology.

Masakazu Kataoka
Shinshu Universiry, Nagano, Japan

Conjugative transfer of bacterial plasmids has been studied from the viewpoint of drug- resistance spreading in the hospital and bacterial genome evolution by the horizontal gene transfer. In addition, the F factor has contributed largely in genetic of Eschericia coli, and Ti plasmid has opened the door of the current plant biotechnology. We believe the horizontal gene transfer using three main mechanisms, transformation, transfection, and conjugation has increased the bacterial genetic diversity. The conjugal transfer has suitable characteristics for synthetic biology since free from the capability to transfer the large DNA without exposing it in water. The huge genome information of various organisms has been accumulated by appearance of the next-generation sequencer and high performance computers. The synthetic biology of designed genome using the huge information must be one of the targets in next generation biology. However, the handling and cloning method of a large DNA fragment are still difficult way, and limited bacterial species can be used for artificial transformation. We have been trying to solve these difficulties by using the conjugal transfer, relatively old knowledge. In this CSHL meeting, I would like to present and discuss our recent three challenges for handling Streptomyces and Bacillus bacteria.

The conjugal transfer for the handling of Streptomyces secondary metabolism.
We use three techniques, the conjugal transfer between the E. coli - Streptomyces using RP4 system, the insertion of the large DNA fragment onto linear plasmid by actinophage system, the conjugal transfer between Streptomyces by the conjugation system of linear Streptomyces plasmid. The established system can be used to improve various biosynthesis of many Streptomyces strains without developing protoplast operation and transformation.

High-frequency transmission of the Streptomyces genome.
We developed the high frequency genome mobilization system in Streptomyces. The system was accomplished by insertion of 4kb fragment coding transfer genes of Streptomyces RCR type small size plasmid named pSN22.

Optimization by the conjugal transfer to Bacillus.
We optimized the inter-genus conjugal transfer between E. coli and B. subtilis using RP4 system. We expect this technique can be applied to strains being closely related to B. subtilis with difficulties of normal transformation including B. natto.

Insights into the Zymomonas mobilis plasmidome

Ersi Emmanouilidou 1 , NikosTaliouras 1 , Valia Tampakopoulou 1 , Karen Davenport 2 , David Bruce 3 , Chris Detter 2 , Roxanne Tapia 2 , Cliff Han 2 , Miriam L. Land 4 , Loren Hauser 4 , Yun-Juan Chang 4 , Chrongle Pan 4 , Vassili N. Kouvelis 1 , Lynne A. Goodwin 2 , Tanja Woyke 2 , Nikos C. Kyrpides 3 , Milton A. Typas 1 , and Katherine M. Pappas 1*
1 Department of Genetics & Biotechnology, Faculty of Biology, University of Athens, Panepistimiopolis, Athens 15701, Greece
2 DOE Joint Genome Institute, Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545
3 DOE Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, California 94598
4 Oak Ridge National Laboratory, Bioscience Division, Oak Ridge, Tennessee 37831
* corresponding: [email protected]

Zymomonas mobilis is a candidate organism for large scale bioethanol production due to its ability to ferment sugars to ethanol faster than yeasts and to higher final yields. In order to understand the biology of Z. mobilis, six different strains belonging to its major subspecies, isolated from various parts of the globe, are being sequenced at the US Department of Energy – Joint Genome Institute in collaboration with the University of Athens (CSP_788284 (DNAseq) CSP_52 (RNAseq) Plasmids of the examined strains have received special attention and have been sequenced twice for each strain: once in terms of WGS sequencing and also from plasmid material provided in separate. All Z. mobilis strains harbor plasmids that range in numbers from two to eight per strain, in sizes from 1.6 kb to 53 kb, and often sum-up to 8% of a strain’s genome. The smallest plasmids are high-copy rolling-circle-replicating and some are mobilizable they are suitable for the generation of species-specific cloning and expression vectors. The larger plasmids are theta-replicating, and are also important for the creation of low-copy vehicles for gene or library introduction. Of the over 600 genes annotated on the sequenced Z. mobilis plasmids, genes of interest contain genes involved in basic metabolism, structure formation, regulation, transposition, immunity (CRISPR) and tolerance to adverse agents, as well as housekeeping genes. Among these last, well discerned are replication, active partitioning and toxin-antitoxin genes. Plenty of the Z. mobilis plasmid genes are strain-specific, while others have homologs in different strains. Apart from instances where distinct syntenic regions are found on plasmids of more than one strain, no particular plasmid seems to have been horizontally dispersed and retained between strains in its entirety. Additionally, and despite the plentiful IS elements harbored in most strains, strain chromosomes do not appear to host traces of plasmid material. In fact, in one strain (NCIMB 11163) an entire conjugative region is met on the chromosome and not on any of its plasmids (or any other strain&aposs plasmids), as a whole or part of. This gives the impression that the plasmidome of Z. mobilis is highly conserved, which is reinforced by the fact that plasmids of different Z. mobilis strains have been used consistently throughout the years for strain-profiling purposes.

Investigating plasmid transfer in environmental matrices using molecular approaches

Xavier Bellanger, Hélène Guilloteau, Christophe Merlin *
Laboratoire de Chimie Physique et Microbiologie pour l&aposEnvironnement, UMR 7564 Université Lorraine - CNRS, 15 avenue du Charmois, 54500 Vandoeuvre-lès-Nancy, Nancy, France
* corresponding: [email protected]

Antibiotic resistance gene transfer mediated by plasmids is a matter of concern for public health, but permissive environments/conditions supporting plasmid dissemination are still quite difficult to identify. Lately, we have developed a molecular approach based on quantitative PCR to monitor the fate of known plasmids in natural microbial communities maintained in microcosms. Practically, it consists in inoculating microcosms with a donor bacterium and monitoring the evolution of both the plasmid and the initial host DNAs over time. Because conjugative transfer is an intercellular form of DNA replication, the plasmid to donor DNA ratio increases in community DNA when the plasmid disseminates by conjugation into the indigenous population. As far as very specific sets of primers and probes are available for the non-ambiguous quantification of donor and plasmid DNAs, this method provides the advantage of considering plasmid transfer in a wide range of possible indigenous recipient bacteria, culturable or not, and it is sensitive enough to detect rare transfer events under low (but realistic) donor inoculum size. Using the broad host range IncP-1&beta plasmid pB10 as model, such transfer experiments were carried out in various environmental matrices from river sediments, to activated sludge, and manure. These experiments demonstrated that the transfer of the conjugative-proficient plasmid pB10 in complex environments is relatively rare and is strongly matrix dependent. The detection of successful transfer events in a given environmental matrix seemed to be linked to the initial stability of the donor inoculum. Depending on the matrix considered, eukaryotic predation plays a significant role in either limiting or promoting the plasmid transfer events. An attempt to link the microbial community structure and the matrix permissiveness showed that TTGE analysis is not resolutive enough to point out common features among comparable communities supporting pB10 transfer. However, an estimation of the IncP-1&alpha/&beta plasmids abundance by quantitative PCR demonstrated that pB10 transfer tends to be supported by environmental matrices exhibiting a higher content of IncP-1&alpha/&beta plasmids. We suggest that the relative abundance of IncP-1 plasmids in a given microbial community reflects its permissiveness to the transfer of plasmids belonging to the same incompatibility group, which prevails over transfer limitation due to the phenomenon known as superinfection immunity.

To conjugate or not to conjugate? The importance of conjugation in the persistence of large plasmids is highly host dependent.

Porse A, Munck C, Sommer MO
Technical University of Denmark

It is not well understood how plasmids persist under non-selective conditions in nature 1 . The first efforts to address this question were carried out in the 1970s through mathematical modelling of plasmid bearing populations 2,3,4 . These first models predicted a broad range of conditions under which conjugative plasmids will persist by means of infectious transfer alone. However, they were often based on parameters obtained in vitro using laboratory strains and plasmids 4,5 . While some suggest high transfer rates to be vital for parasitic plasmid survival, others argue that the transfer rates predicted to be necessary are not attainable, nor necessary, for plasmid persistence in natue 6,7,8,9 . Consequently, the importance of conjugation in the persistence of plasmids has been heavily debated in the plasmid community 6,8,10 .

We have investigated the genetic changes implicated in host-plasmid adaptation between a clinical ESBL plasmid and different clinically relevant E. coli and K. pneumoniae hosts. Using adaptive evolution and subsequent population sequencing, we show that the burden imposed on naïve cells receiving a conjugative plasmid is mainly caused by the conjugational transfer machinery itself and varies substantially between hosts. We suggest that variations in the ability to regulate genes involved in conjugation is the major cause of the observed differences in plasmid cost, ultimately resulting in deletion of non-regulated genes. These observations highlight the trade-off between horizontal and vertical transfer and indicate that the fitness cost, having to be compensated by the conjugational machinery in order for the plasmid to persist, is highly dependent on the host background and that the conjugational machinery can be strongly disfavoured in certain hosts. Our findings add an extra layer of complexity to the ongoing discussion concerning the importance of conjugational transfer for plasmid persistence in diverse bacterial populations.

1 Harrison, E. & Brockhurst, M. a. Plasmid-mediated horizontal gene transfer is a coevolutionary process. Trends Microbiol. 20, 262–7 (2012).
2 Stewart, F. & Levin, B. The population biology of bacterial plasmids: a priori conditions for the existence of conjugationally transmitted factors. Genetics 209–228 (1977).
3 Levin, B. R. & Stewart, F. M. Probability of establishing chimeric plasmids in natural populations of bacteria. Science 196, 218–20 (1977).
5 Slater, F. R., Bailey, M. J., Tett, A. J. & Turner, S. L. Progress towards understanding the fate of plasmids in bacterial communities. FEMS Microbiol. Ecol. 66, 3–13 (2008).
6 Lili, L. N., Britton, N. F. & Feil, E. J. The persistence of parasitic plasmids. Genetics 177, 399–405 (2007).
7 Bergstrom, C. T., Lipsitch, M. & Levin, B. R. Natural selection, infectious transfer and the existence conditions for bacterial plasmids. Genetics 155, 1505–19 (2000).
8 Freter, R., Freter, R. R. & Brickner, H. Experimental and mathematical models of Escherichia coli plasmid transfer in vitro and in vivo. Infect. Immun. 39, 60–84 (1983).
9 Imran, M., Jones, D. & Smith, H. Biofilms and the plasmid maintenance question. Math. Biosci. 193, 183–204 (2005).
10 Tazzyman, S. J. & Bonhoeffer, S. Fixation probability of mobile genetic elements such as plasmids. Theor. Popul. Biol. 90, 49–55 (2013).

Plasmids help bacteria to survive stress

Plasmids contain just a few genes, but they make a big difference to their host bacterium. The genes are usually not essential for the bacterium’s day-to-day survival – instead, they help the bacterium to overcome occasional stressful situations. For instance, many plasmids contain genes that, when expressed, make the host bacterium resistant to an antibiotic (so it won’t die when treated with that antibiotic). Other plasmids contain genes that help the host to digest unusual substances or to kill other types of bacteria.

Col Plasmids

Col plasmids confer to bacteria the ability to produce toxic proteins known as colicines. Such bacteria as E. coli, Shigella and Salmonella use these toxins to kill other bacteria and thus thrive in their respective environments.

There are different types of Col plasmids in existence that produce different types of colicines/colicins. A few examples of Col plasmids include Col B, Col E2 and E3. Their differences are also characterized by differences in their mode of action.

For instance, whereas Col B causes damage to cell membrane of other bacteria (lacking the plasmid) Col E3 has been shown to induce degradation of the nucleic acids of the target cells.

Like fertility plasmids, some of the Col plasmids have been shown to carry elements that enhance their transmission from one cell to another. Therefore, through conjugation or the mating process, particularly for cells with the F factor (fertility plasmids) the Col plasmids can be transferred from one cell (donor) to another (recipient).

As a result, the recipient acquires the ability to produce toxins that kill or inhibit the growth of the target bacteria lacking the plasmid.

* Colicins/colicines belong to a group of toxins known as bacteriocins.

* These toxins affect the target bacteria by affecting such processes as replication of DNA, translation and energy metabolism among others.

Where do plasmids come from? And why are they there?

So recently we talked about plasmids in my biology class and the first thought that popped into my head was why is there something so useful just laying around waiting for a bacteria to pick it up? I mean in the context of all living things this seems to make the least sense. Generally speaking nature is random. Mutations are random and sometimes are helpful but most of the time harmful. So why would there be something so beneficial just out there? Forgive me if i have something incorrect, I am only a business major so biology isn't exactly my forte.

It is probable that plasmids evolved within bacteria, separately from the primary bacterial chromosome (the main unit of inheritance within bacteria).

Plasmids carry genes. For example, a gene coding for antibiotic resistance. As they can be transferred between bacteria, (the chromosome cannot) it is more likely that bacteria containing these plasmids will survive to reproduce, and to further spread the plasmid via a process called conjugation.

Plasmids probably didn't evolve just like 'that', they probably came into existence through a series of random mutations - which is the basic principle of evolution - a slow accumulation of random mutations that are beneficial to the host. The fact that most are harmful are the reason why this process is so slow. (That and DNA proofreading etc etc).

Final year Molecular Bio undergrad here. References and verification can be given on request.

Also, please let me know if you need further clarification, I'm aware that wasn't worded amazingly.

Wild plasmids occur naturally like mini-chromosomes and can carry genes that help a bacterial population survive. They come with origins of replication and copy-number control mechanisms.

The biotech plasmids that you hear about in freshman college classes are engineered versions, sometimes using building blocks from several different wild plasmids. Start with the origin of replication of one, paste in antibiotic resistance of another to be able to select for bacteria that carry the plasmid, then paste in a promoter and terminator and all the other machinery it takes to get a gene expressed (often from the chromosome or a virus/bacteriophage). And finally, add in an entirely artificial "multiple cloning site" or string of useful restriction enzyme cut sites so you can splice in your favorite gene.

Plasmids can be "selfish DNA elements" along with transposons and other DNA sequences that seem to exist simply to replicate its own sequences. The fact that bacterial plasmids can carry antibiotic resistance genes is also selfish if the host cell can cope with the environment better, more copies of the plasmid will be made. So in thinking about your question, look at the problem form the plasmid's point of view, not the cell's.

For instance, there is the postsegregational killing system (PSK) where a plasmid produces a poison that last a long time, and an antidote that lasts a short time. When a cell divides, if one daughter cell doesn't get the plasmid, the poison present in the cytoplasm will kill it. So the plasmid is killing any cells that don't carry it. You can read more about the PSK system on the R1 plasmid in E. coli on Wikipedia.

If you (or anyone reading this comment) is really interested in the origin of plasmids, you might check out Origin and Evolution of Plasmids by Clarence I. Kado (Antonie van Leeuwenhoek January 1998, Volume 73, Issue 1, pp 117-126) PDF available

Isolating DNA

The first step in making recombinant DNA is to isolate donor and vector DNA. General protocols for DNA isolation were available many decades before the advent of recombinant DNA technology. With the use of such methods, the bulk of DNA extracted from the donor will be nuclear genomic DNA in eukaryotes or the main genomic DNA in prokaryotes these types are generally the ones required for analysis. The procedure used for obtaining vector DNA depends on the nature of the vector. Bacterial plasmids are commonly used vectors, and these plasmids must be purified away from the bacterial genomic DNA. A protocol for extracting plasmid DNA by ultracentrifugation is summarized in Figure 12-2. Plasmid DNA forms a distinct band after ultracentrifugation in a cesium chloride density gradient containing ethidium bromide. The plasmid band is collected by punching a hole in the plastic centrifuge tube. Another protocol relies on the observation that, at a specific alkaline pH, bacterial genomic DNA denatures but plasmids do not. Subsequent neutralization precipitates the genomic DNA, but plasmids stay in solution. Phages such as λ also can be used as vectors for cloning DNA in bacterial systems. Phage DNA is isolated from a pure suspension of phages recovered from a phage lysate.

Figure 12-2

Plasmids such as those carrying genes for resistance to the antibiotic tetracycline (top left) can be separated from the bacterial chromosomal DNA. Because differential binding of ethidium bromide by the two DNA species makes the circular plasmid DNA (more. )


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Smillie, C., Garcillán-Barcia, M. P., Francia, M. V., Rocha, E. P. C. & de la Cruz, F. Mobility of plasmids. Microbiol. Mol. Biol. Rev. 74, 434–452 (2010). A thorough analysis of plasmid mobility that sets the basis for mobility typing.

Douarre, P.-E., Mallet, L., Radomski, N., Felten, A. & Mistou, M.-Y. Analysis of COMPASS, a new comprehensive plasmid database revealed prevalence of multireplicon and extensive diversity of IncF plasmids. Front. Microbiol. 11, 1–15 (2020).

Acman, M., van Dorp, L., Santini, J. M. & Balloux, F. Large-scale network analysis captures biological features of bacterial plasmids. Nat. Commun. 11, 2452 (2020). A comprehensive analysis of plasmid whole-genome sequences using similarity networks that paves the way for novel plasmid classification schemes.

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During transformation, in genetic cloning using bacteria plasmids as vectors, do bacteria take in the new plasmid while their plasmid exits?

The bacteria used in cloning already have plasmids from my understanding. And when we insert a genetically altered plasmid, say for insulin production, and we instigate a transformation reaction the bacteria takes in the new plasmid. What happens to their old, original plasmid? It would be odd if they had two distinct plasmids, or at least I would guess it would be inefficient at producing insulin(in this case)

Why do you use wild type e.coli? I thought labs tended to just use mutants

Not all bacteria naturally contain plasmids. But I think you are more interested to know if bacteria can maintain multiple plasmids. The short answer is yes. Multiple plasmids can be maintained as long as there is selective pressure and the origin of replication on two plasmids are compatible.

In a lab selective pressure is generated by design plasmids with antibiotic resistance genes in addition to gene of interest (such as the insulin example of yours). So when you transform both plasmids and grow the cells on a media containing two corresponding antibiotics, only bacteria with both plasmids can survive. In nature selective pressure can be more complex based on the competition and available food sources.

Origin of replication is also important because plasmids are replicated by a process known as rolling circle replication. if two origin of replication sites are too similar, replication might fail due to two plasmids fusing at origin of replication sites.

Watch the video: Origin of Replication - Plasmids 101 (May 2022).


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