What do genetic engineers use plasmids for

It is commonly used in protein engineering. The distribution of fitness effects : The distribution of fitness effects of mutations in vesicular stomatitis virus.

In this experiment, random mutations were introduced into the virus by site-directed mutagenesis. The fitness of each mutant was compared with the ancestral type. A fitness of zero, less than one, one, more than one, respectively, indicates that mutations are lethal, deleterious, neutral, and advantageous.

The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest. The mutation may be a single base change a point mutation , multiple base changes, deletion, or insertion. The single-stranded primer is then extended using a DNA polymerase, which copies the rest of the gene.

The copied gene contains the mutated site. It is then introduced into a host cell as a vector and cloned. Finally, mutants are selected. The original method using single-primer extension was inefficient due to a lower yield of mutants.

The resulting mixture may contain both the original unmutated template as well as the mutant strand, producing a mix population of mutant and non-mutant progenies. The mutants may also be counter-selected due to presence of a mismatch repair system which favors the methylated template DNA.

Many approaches have since been developed to improve the efficiency of mutagenesis. Reproductive cloning, possible through artificially-induced asexual reproduction, is a method used to make a clone of an entire organism. Reproductive cloning is a method used to make a clone or an identical copy of an entire multicellular organism. Most multicellular organisms undergo reproduction by sexual means, which involves genetic hybridization of two individuals parents , making it impossible to generate an identical copy or clone of either parent.

Recent advances in biotechnology have made it possible to artificially induce asexual reproduction of mammals in the laboratory. Reproductive Cloning of Dolly, the Sheep : Dolly the sheep was the first mammal to be cloned. To create Dolly, the nucleus was removed from a donor egg cell. The nucleus from a second sheep was then introduced into the cell, which was allowed to divide to the blastocyst stage before being implanted in a surrogate mother.

An example of parthenogenesis occurs in species in which the female lays an egg. If the egg is fertilized, it is a diploid egg and the individual develops into a female; if the egg is not fertilized, it remains a haploid egg and develops into a male.

The unfertilized egg is called a parthenogenic, or virgin, egg. Some insects and reptiles lay parthenogenic eggs that can develop into adults. Sexual reproduction requires two cells; when the haploid egg and sperm cells fuse, a diploid zygote results. The zygote nucleus contains the genetic information to produce a new individual. However, early embryonic development requires the cytoplasmic material contained in the egg cell. This idea forms the basis for reproductive cloning.

If the haploid nucleus of an egg cell is replaced with a diploid nucleus from the cell of any individual of the same species called a donor , it will become a zygote that is genetically identical to the donor.

Somatic cell nuclear transfer is the technique of transferring a diploid nucleus into an enucleated egg. It can be used for either therapeutic cloning or reproductive cloning. The first cloned animal was Dolly, a sheep who was born in The success rate of reproductive cloning at the time was very low. Dolly lived for seven years and died of respiratory complications.

There is speculation that because the cell DNA belongs to an older individual, the age of the DNA may affect the life expectancy of a cloned individual. Since Dolly, several animals e. There have been attempts at producing cloned human embryos as sources of embryonic stem cells.

Sometimes referred to as cloning for therapeutic purposes, the technique produces stem cells that attempt to remedy detrimental diseases or defects unlike reproductive cloning, which aims to reproduce an organism. Still, therapeutic cloning efforts have met with resistance because of bioethical considerations. Basic techniques used in genetic material manipulation include extraction, gel electrophoresis, PCR, and blotting methods.

To understand the basic techniques used to work with nucleic acids, remember that nucleic acids are macromolecules made of nucleotides a sugar, a phosphate, and a nitrogenous base linked by phosphodiester bonds. The phosphate groups on these molecules each have a net negative charge. An entire set of DNA molecules in the nucleus is called the genome. DNA has two complementary strands linked by hydrogen bonds between the paired bases.

The two strands can be separated by exposure to high temperatures DNA denaturation and can be reannealed by cooling. This can be done through various techniques. Most nucleic acid extraction techniques involve steps to break open the cell and use enzymatic reactions to destroy all macromolecules that are not desired such as degradation of unwanted molecules and separation from the DNA sample.

Macromolecules are inactivated using enzymes such as proteases that break down proteins, and ribonucleases RNAses that break down RNA. The DNA is then precipitated using alcohol. Human genomic DNA is usually visible as a gelatinous, white mass. RNA analysis is performed to study gene expression patterns in cells. Bacteria are rather simple organisms. They are unicellular, meaning only composed of one cell.

They also have a single chromosome, meaning most of their genes is contained in a single DNA molecule. Well, on top of having a bacterial chromosome, bacteria may also contain plasmids. Those are smaller circle of DNA that have the ability to replicate copy themselves independently of the chromosome. A single bacteria may contain several copies, sometimes hundreds, of a given plasmid. Plasmids also contain additional genes that may help the bacteria to adapt to harsher life conditions.

Because of their size, they are easy to pass on to other bacteria of the same species this is called bacterial conjugation , thus participating to the survival ability of the bacterial strain.

Discovered in [1] , plasmids were rapidly turned into molecular tools to first amplify, then transfer DNA material into other biological systems.

This was possible thanks to the concomitant discovery of both restriction enzymes and DNA ligases during the Sixties. Restriction enzymes constitute a now wide family of proteins able to cut DNA molecules at specific sites defined by their nucleotide sequences. So, by the end of the Sixties, scientists knew of the genetic code and had an idea of the multitude of texts Genomes that were written everywhere in DNA molecules.

Then started a period of genetic scrapbooking to empirically engineer the first DNA vectors. Physiological Reviews. CiteSeerX PMID Despite tremendous progress in a mere 50 years period, the molecular tools the scientific community can rely on are far from fitting all the experimental needs. Indeed, as knowledge increase and questions complexify, the needs for more sophisticated vectors to be designed and produced is challenging the field daily.

Home Discover e-Zyvec Public Close. Linkedin-in Icon-researchgate. Plasmids : essential tools for genetic engineering. Such microbes will contain new sequences of DNA some unintentionally inserted also including desired and seemingly harmless sequences. This may pose a potential threat to the health of plants and animals, including humans, and to other features in the biosphere, especially if they show better growth in the recipient environment when compared to the indigenous microbiota or the experimental parental strains.

Given that, even minor alterations in a single biosynthetic capability can significantly augment growth rates and hence, result in greater survival and colonization by introduced microbes. For instance, can bacteria possessing an acquired N2 fixing ability combined with existing rapid growth, efficient metabolism, and high survival value in natural habitats be able to decrease the atmospheric N2 content, ground-waters polluted with NO3, and deplete the ozone layer due to production of NO x , from NO 3?

Can organisms designed to remove oil-spills remain confined to such spills or will they extend to other areas and cause degradation of petroleum products in the gas station and refinery, especially if genes, which greatly augment their survival ability in these habitats were also acquired? However, there have been few studies investigating the survival of these microbes in natural environments.

Their survival may be vastly improved by an ability of weakened recipients to obtain the genes present in the natural habitat into which they are placed which reduces their intensity of auxotrophy. To date, there are no studies investigating influence of the physicochemical features of the recipient environment on the microbial ability to survive and acquire genes.

These characteristics play a major role in concluding the survival, establishment, and growth of the indigenous as well as the introduced microorganisms in natural habitats [ 23 ]. Genetic recombination studies in bacteria are mostly performed in vitro, and there is limited data proving an in-situ form of gene transfer. A few studies have been conducted in vivo using axenic animals or animals with the normal biota, usually of the intestinal tract, being significantly decreased or completely eliminated by pretreatment with antibiotic.

These studies focused on conjugation, mainly R-factor transfer, as the gene transfer mechanism [ 23 ]. A demonstration of R-factor genes transfer by transduction is seen in Staphylococcus aureus and in Pseudomonas aeruginosa.

Some soil-borne bacteria e. Moreover, a demonstration of the conjugation in soil-borne bacteria in vitro such as pseudomonads is seen [ 23 ]. Increase in nosocomial infections by drug-resistant bacteria counts as empirical evidence, which implies that the gene transfer responsible for antibiotics and heavy metals resistance occurs in natural habitats. However there is few experimental support to make a solid conclusion, as most of these studies have been restricted to either using resistant bacteria isolated from natural habitats or performing the transfer and expression of these genetic materials under carefully measured laboratory conditions.

Essentially no studies have endeavored to connect these experimental extremes, probably due to lack of techniques specific to studying genetic recombination occurring in natural habitats and lack of scientists trained in microbial genetics [ 23 ]. The ability for survival, multiplication, and conjugation in sterile soils is demonstrated in both the strains of E. Clay minerals present, especially montmorillonite, increased the frequency of recombination, most probably due to the enhancement in bacterial growth ability which clay possesses.

Numerous mechanisms, which clarify the way in which clay minerals influence the survival, growth, establishment, and metabolic activities of microbes in natural habitats, have been outlined.

Initial studies on conjugation occurring in nonsterile soils have suggested that the recombination frequency is significantly lower than in sterile soils [ 23 ]. The decreased occurrence of recombination occurring in nonsterile soils confirms the obtained results with the drug-resistance plasmids transfer to an animal system.

The transfer frequency of a multiple drug resistant plasmid from Salmonella typhosa to E. However, a significantly lower frequency of transfer was seen in the presence of other bacteria namely exogens such as Proteus mirabilis and nonconjugative E. This observed reduction was not a consequence of the exogens interfering physically i.

This is demonstrated by polystyrene latex particles with the same size and concentration as that of the exogens not affecting the frequency of plasmid transfer, suggesting a chemical interference on conjugation caused by the exogens.

It is unknown whether the lower frequencies of conjugation in nonsterile compared to sterile soils are attributed to such interference, but that interference could be possible since various species may be in adjacent vicinity in different natural microbial habitats [ 23 ].

Studies involving the conjugation in sterile soil also signified that rather than undergoing genetic recombination, cross-feeding syntrophism allows bacteria that are auxotrophic for various nutrients to co-exist, in both soil and replica-plated agar media.

This observation accentuates the need to prudently investigate suggestions for seeming genetic recombination occurring in natural habitats and the ability of auxotrophs to survive in natural habitats as a viable possibility, in spite of their apparent fragility and debilitation, in the chance that other microbes present in the same habitat act as commensals providing nutrients which cannot be synthesized by auxotrophs.

Sagik and Sorber showed that these auxotrophs e. The solid fraction of the waste stream appears to be associated with this survival, once again demonstrating that particulates and the resultant increases in surface area improve the survival and growth of bacteria. There is insufficient documentation shedding light on transformation, which occurs in natural microbial habitats.

Greaves and Wilson have, however, demonstrated that nucleic acids become adsorbed to soil clay minerals, particularly to montmorillonite, and that the adsorption protects the nucleic acids from degradation by enzymes.

Similarly clay adsorbed viruses, proteins, peptides, and amino acids are protected to different degrees against microbial degradation. Accordingly, both naked DNA taking part in transduction may endure in natural habitats despite the absence of an appropriate host [ 23 ].

This adsorption to clay minerals protecting soluble organics and viruses from degradation is vital to consider in any possible exchange of genes occurring in clay containing habitats and other surface-active particulates.

An inability of transforming DNA and transducing viruses to survive can be expected for long in natural habitats lacking hosts. In addition, being best, the substrate for nonhost microbes that is they contain C, N, and P as well as S in case of viruses means they would be swiftly degraded by the indigenous microbiota. However, there is growing evidence that DNA and viruses persevere in natural habitats due to the clay minerals adsorption process, which protects against both biological inactivation and physico-chemical.

Thus, if transforming DNA and viruses no studies have investigated the ability of adsorbed DNA to transform are able to persist in natural habitats, it is possible that it is through transmission of their genetic information to any suitable host introduced into these habitats inadvertently or deliberately.

There are sporadic studies involving the survival and consequent microbial establishment of microbes, which do not inhabit a particular habitat. This is illustrated by the survival ability of enteric bacteria including E.

It cannot be said that plasmids are mere materials and a suitable environment for genetic exchange given that they themselves are subjects to evolutionary forces [ 24 ]. The connections of plasmid access in new bacteria result in a cost of fitness.

Therefore, if the plasmid is unable to spread horizontally with the required speed ensuring its survival as a pure gene parasite, the theory predicts that it will be removed from the bacterial group. Thus, unless the plasmid-encoded traits are not selected, the plasmid of the population will be removed by purifying the selection. Furthermore, positive selections can ultimately result in any beneficial plasma genes to move to the bacterial chromosome; hence, the beneficial value of the plasmid is elminiated.

However, the costs inflicted by plasmids are not irreversible. In fact, the latest reports indicate that compensatory development can often improve these costs. Extensive studies conducted by clinical microbiologists have shed light on the molecular basis and epidemiology of AR, giving a clear perception of which genes and AR plasmids proliferate in clinical conditions.

In this process, the plasmid bacteria acquire genetic charge that can provide feature. Arrows indicate the directions in which the genes are transcribed. Note the polylinker site, containing multiple unique restriction enzyme recognition sites, found within the lacZ reporter gene. Also note the ampicillin amp resistance gene encoded on the plasmid. Plasmid vectors used for cloning typically have a polylinker site , or multiple cloning site MCS.

A polylinker site is a short sequence containing multiple unique restriction enzyme recognition sites that are used for inserting DNA into the plasmid after restriction digestion of both the DNA and the plasmid. Having these multiple restriction enzyme recognition sites within the polylinker site makes the plasmid vector versatile, so it can be used for many different cloning experiments involving different restriction enzymes. This polylinker site is often found within a reporter gene , another gene sequence artificially engineered into the plasmid that encodes a protein that allows for visualization of DNA insertion.

The reporter gene allows a researcher to distinguish host cells that contain recombinant plasmids with cloned DNA fragments from host cells that only contain the non-recombinant plasmid vector.

The most common reporter gene used in plasmid vectors is the bacterial lacZ gene encoding beta-galactosidase, an enzyme that naturally degrades lactose but can also degrade a colorless synthetic analog X-gal , thereby producing blue colonies on X-gal—containing media.

The lacZ reporter gene is disabled when the recombinant DNA is spliced into the plasmid. Because the LacZ protein is not produced when the gene is disabled, X-gal is not degraded and white colonies are produced, which can then be isolated. This blue-white screening method is described later and shown in Figure 4. In addition to these features, some plasmids come pre-digested and with an enzyme linked to the linearized plasmid to aid in ligation after the insertion of foreign DNA fragments.

Figure 4. Click for a larger image. The steps involved in molecular cloning using bacterial transformation are outlined in this graphic flowchart. The most commonly used mechanism for introducing engineered plasmids into a bacterial cell is transformation , a process in which bacteria take up free DNA from their surroundings. Some bacteria, such as Bacillus spp. However, not all bacteria are naturally competent. In most cases, bacteria must be made artificially competent in the laboratory by increasing the permeability of the cell membrane.

This can be achieved through chemical treatments that neutralize charges on the cell membrane or by exposing the bacteria to an electric field that creates microscopic pores in the cell membrane.

These methods yield chemically competent or electrocompetent bacteria, respectively. Following the transformation protocol, bacterial cells are plated onto an antibiotic-containing medium to inhibit the growth of the many host cells that were not transformed by the plasmid conferring antibiotic resistance. A technique called blue-white screening is then used for lacZ -encoding plasmid vectors such as pUC Blue colonies have a functional beta-galactosidase enzyme because the lacZ gene is uninterrupted, with no foreign DNA inserted into the polylinker site.

These colonies typically result from the digested, linearized plasmid religating to itself. However, white colonies lack a functional beta-galactosidase enzyme, indicating the insertion of foreign DNA within the polylinker site of the plasmid vector, thus disrupting the lacZ gene. Thus, white colonies resulting from this blue-white screening contain plasmids with an insert and can be further screened to characterize the foreign DNA.

The bacterial process of conjugation see How Asexual Prokaryotes Achieve Genetic Diversity can also be manipulated for molecular cloning. F plasmids , or fertility plasmids, are transferred between bacterial cells through the process of conjugation.

Recombinant DNA can be transferred by conjugation when bacterial cells containing a recombinant F plasmid are mixed with compatible bacterial cells lacking the plasmid. F plasmids encode a surface structure called an F pilus that facilitates contact between a cell containing an F plasmid and one without an F plasmid. On contact, a cytoplasmic bridge forms between the two cells and the F-plasmid-containing cell replicates its plasmid, transferring a copy of the recombinant F plasmid to the recipient cell.

Once it has received the recombinant F plasmid, the recipient cell can produce its own F pilus and facilitate transfer of the recombinant F plasmid to an additional cell.

The use of conjugation to transfer recombinant F plasmids to recipient cells is another effective way to introduce recombinant DNA molecules into host cells. Alternatively, bacteriophages can be used to introduce recombinant DNA into host bacterial cells through a manipulation of the transduction process see How Asexual Prokaryotes Achieve Genetic Diversity. In the laboratory, DNA fragments of interest can be engineered into phagemids , which are plasmids that have phage sequences that allow them to be packaged into bacteriophages.

Bacterial cells can then be infected with these bacteriophages so that the recombinant phagemids can be introduced into the bacterial cells. Molecular cloning may also be used to generate a genomic library. Having such a library allows a researcher to create large quantities of each fragment by growing the bacterial host for that fragment. These fragments can be used to determine the sequence of the DNA and the function of any genes present.

One method for generating a genomic library is to ligate individual restriction enzyme-digested genomic fragments into plasmid vectors cut with the same restriction enzyme Figure 5. After transformation into a bacterial host, each transformed bacterial cell takes up a single recombinant plasmid and grows into a colony of cells. All of the cells in this colony are identical clones and carry the same recombinant plasmid.