Name: antibiotic resistance in cloning
Uploaded: 2026-08-26T08:20:54.022Z
Duration: 1 min 45 s
Channel: JoVE

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From Molecules To Organisms: Structures And Processes

HS-LS1-8

Antibiotic Selection

Antibiotic selection is a method used to identify bacterial cells that have taken up a plasmid.A plasmid is a small circular DNA molecule that includes a gene of interest for expression in host cells, along with an antibiotic resistance gene that provides resistance to specific antibiotics.During transformation, plasmid DNA and bacterial cells are incubated together. A heat shock step is then performed, causing some cells to take up the plasmid, while many cells do not receive the DNA.After a brief recovery period, the cells are plated onto a medium containing the selected antibiotic.Transformed cells with the resistance gene produce proteins that break down the antibiotic or block its effects. This allows the cells to survive and grow.Non-transformed cells do not contain the resistance gene, so they cannot survive on the antibiotic-containing medium.Because the resistance gene and the gene of interest are located on the same plasmid, the surviving cells usually also contain the gene of interest.Following antibiotic selection, only transformed cells carrying the recombinant plasmid survive and form visible bacterial colonies on the plate.After selection, scientists pick these colonies and screen them using methods such as colony PCR, restriction digestion, or DNA sequencing to confirm the presence of the gene of interest.

antibiotic selection

Researchers use antibiotic resistance genes to identify bacteria that possess a plasmid containing their gene of interest. Antibiotic resistance naturally occurs when a spontaneous DNA mutation creates changes in bacterial genes that eliminate antibiotic activity. Bacteria can share these new resistance genes with their offspring and other bacteria. The overuse and misuse of antibiotics have created a public health crisis, as resistant and multi-resistant bacteria continue to develop.Antibiotic Resistance is an Essential Tool in Genetic EngineeringAntibiotics, such as penicillin, are drugs that kill or stop bacterial growth. Bacteria that naturally or artificially acquired antibiotic resistance genes do not respond to antibiotics. Scientists exploit this by designing plasmids&#x2014;small, self-replicating pieces of DNA&#x2014;that carry both an antibiotic resistance gene and a gene of interest. Antibiotic resistance is an integral part of DNA cloning that allows a researcher to identify cells that absorbed a DNA of interest.The researcher&#x2019;s DNA of interest is introduced into bacterial cells using a process called transformation. Bacterial transformation involves temporarily creating small holes in the bacterial cell wall to permit the uptake of external DNA such as a plasmid. Only some bacterial cells absorb new DNA. Since the plasmid includes both the DNA of interest and a gene that confers resistance to a specific antibiotic, applying the antibiotic to the bacterial cells (i.e., antibiotic selection) can help determine which cells were genetically modified.The researcher spreads the bacterial cells onto a culture plate containing a chosen antibiotic. Only bacteria containing the antibiotic resistance gene survive and grow on the plate. After a few days, the researcher can select a bacterial colony to culture for further experiments&#x2014;such as gene expression studies. Following antibiotic selection, the researcher will further test the bacteria using other methods (e.g., PCR) to confirm that DNA of interest is correct. Errors often occur such as the plasmid not containing the gene of interest at all.Bacteria Can Acquire Antibiotic Resistance NaturallyBacteria can acquire antibiotic resistance through spontaneous DNA mutations that alter the proteins produced by the cell. Resistant bacteria may produce proteins that cause the antibiotic to either be degraded, pumped out of the cell, or prevented from interacting with its target. For example, the antibiotic vancomycin inhibits synthesis of the bacterial cell wall. Some bacteria have developed resistance to this antibiotic by changing the types of protein subunits&#x2014;amino acids&#x2014;used in the assembly of their cell wall to ones that are unaffected by vancomycin.Once antibiotic resistance genes emerge, bacteria can pass them on to their offspring. Bacteria can also acquire antibiotic resistance genes from other bacteria of the same or different species through a process called horizontal gene transfer (HGT). There are three mechanisms of HGT: transformation, conjugation, and transduction. Antibiotic resistance genes are often found in plasmids or transposons&#x2014;pieces of DNA that are easily transferred between bacteria&#x2014;that are exchanged during HGT. As a result, new types of antibiotic resistance can rapidly spread to multiple types of infectious bacteria.Clinical Overuse and Misuse of Antibiotics Produces &#x201C;Superbugs&#x201D;Antibiotics are a critical treatment for bacterial infections. However, their use can cause bacteria to become resistant and render the antibiotic ineffective, leading to untreatable and potentially deadly infections. Antibiotic overuse and misuse&#x2014;for instance, using antibiotics to treat viral (rather than bacterial) infections or to increase livestock growth&#x2014;is problematic because it promotes resistance.Antibiotics cause resistance to evolve because they kill susceptible bacteria and leave only the resistant individuals. The surviving bacteria divide rapidly, producing offspring with the same antibiotic resistance. When antibiotics are overused, this selection pressure causes the number of resistant bacteria in the population to rise quickly. This is a major public health concern because it increases antibiotic resistance and creates &#x201C;superbugs&#x201D; that are resistant to multiple antibiotics. Continued overuse and misuse of antibiotics may eventually exhaust treatment options for bacterial infections.

Overview

Researchers use antibiotic resistance genes to identify bacteria that possess a plasmid containing their gene of interest. Antibiotic resistance ...

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Biology

Genetics

Biotechnology

KEY TERMS AND CONCEPTS

antibiotic resistance in cloning

Antibiotic resistance genes help scientists find bacteria that took up a plasmid with a gene of interest. In genetic engineering, this resistance is used as a marker, not just as a medical concern. The same process also connects to the growing problem of resistant bacteria in the real world.

Antibiotics, such as penicillin, stop bacterial growth or kill bacteria. When bacteria naturally have, or are engineered to carry, an antibiotic resistance gene, the antibiotic no longer works on them. Researchers build plasmids, which are small pieces of self-replicating DNA, with both the gene of interest and a resistance gene. This makes antibiotic resistance an important part of DNA cloning and cell selection.

To move DNA into bacteria, scientists use transformation. During transformation, small holes are made in the bacterial cell wall so external DNA, like a plasmid, can enter. Only some cells take up the new DNA. Scientists then place the bacteria on a plate containing the chosen antibiotic. Only cells with the resistance gene survive and form colonies.

After a few days, a colony can be picked and grown for later experiments, such as gene expression studies. The bacteria are often tested again with methods like PCR to confirm that the DNA of interest is correct. This step matters because mistakes can happen, including plasmids that do not contain the target gene at all.

Bacteria can also gain antibiotic resistance naturally through spontaneous DNA mutations. These changes may produce proteins that break down the antibiotic, pump it out of the cell, or block it from reaching its target. For example, vancomycin blocks bacterial cell wall synthesis, but some bacteria avoid its effect by changing the amino acid subunits used to build their cell wall.

Once resistance genes appear, bacteria can pass them to offspring. They can also share them with other bacteria through horizontal gene transfer, or HGT. The three main HGT methods are transformation, conjugation, and transduction. Resistance genes are often carried on plasmids or transposons, which can move easily between cells and spread new resistance traits quickly.

Antibiotic overuse and misuse have made this problem worse. Using antibiotics for viral infections or to increase livestock growth can select for resistant bacteria. The susceptible bacteria die, while resistant survivors keep dividing and passing on the same trait. Over time, this can lead to multi-resistant bacteria, or superbugs, and may leave fewer treatment options for bacterial infections.

Antibiotic Resistance in Cloning

what is genetic engineering?

How Scientists Edit DNA for New Traits

Genetic engineering changes an organism’s DNA to add useful traits. Scientists use it in bacteria, plants, animals, and even viruses for research, medicine, agriculture, and industry. It can bring major benefits, but it also raises ethical concerns about humans and the food supply. This work is possible because DNA has the same basic structure and genetic code across life. That shared code lets scientists modify DNA in several ways. One method is CRISPR/Cas9, often called molecular scissors,...

dna isolation

Steps in DNA Extraction

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recombinant dna technology

Recombinant DNA in Cloning and GMOs

Recombinant DNA is made by joining DNA from different sources in the lab. Scientists often use it to clone genes and study them in cells that are easy to handle, such as bacteria. Organisms that carry recombinant DNA are called genetically modified organisms, or GMOs. This technology gives scientists new tools for research, medicine, and agriculture. To build recombinant DNA, researchers place a gene of interest into a vector, which is a DNA carrier that moves foreign DNA into a host cell. The...

PCR Cycle for DNA Amplification

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complementary dna

From mRNA to cDNA for Gene Studies

Complementary DNA, or cDNA, is made from messenger RNA (mRNA) so scientists can study which genes are active in cells and tissues. Only genes that are transcribed into mRNA are expressed. By extracting mRNA from cells, researchers can compare gene expression across samples. cDNA is produced by reverse transcription. This process copies mRNA into DNA, which is useful because mRNA does not contain introns, or non-coding regions, and other regulatory sequences. As a result, cDNA can help...

Antibiotic resistance genes help scientists find bacteria that took up a plasmid with a gene of interest. In genetic engineering, this resistance is used as a marker, not just as a medical concern. The same process also connects to the growing problem of resistant bacteria in the real world. Antibiotics, such as penicillin, stop bacterial growth or kill bacteria. When bacteria naturally have, or are engineered to carry, an antibiotic resistance gene, the antibiotic no longer works on them.

transgenic organisms

Creating Transgenic Plants and Animals

Transgenic organisms are genetically engineered to carry a transgene, which is a gene from a different species, in their genome. The inserted gene may be a new gene for that organism or a different version of one it already has. Scientists usually make transgenes with recombinant DNA and DNA cloning methods. Researchers use transgenic bacteria, plants, and animals to answer biological questions and to build useful tools. To make a transgenic organism, they first choose a transgenesis method,...

in-vitro mutagenesis (gene knockouts)

Knockout Mice and Gene Replacement

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crispr

CRISPR-Cas9 Gene Editing in Action

CRISPR-Cas9 is a genome editing tool that lets scientists change DNA at a specific location. It can add, remove, or rearrange genetic material. Researchers study it because it may help treat genetic disorders such as hemophilia and sickle cell anemia. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, and Cas9 is a CRISPR-associated protein 9. The basic system includes a Cas9 endonuclease, which is an enzyme that cuts DNA, and a small RNA that guides Cas9 to the...

gene therapy

Gene Delivery for Serious Diseases

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embryonic stem cells

How Embryonic Stem Cells Become Specialized

Embryonic stem cells are pluripotent cells that have not yet specialized. Pluripotent means they can give rise to many different cell types in the body. Because of this, they are useful in science and medicine for making cells needed in research or for replacing cells lost to damage or disease. These cells come from the inner cell mass of a blastocyst, which is an early embryo about 3 to 5 days after fertilization in humans. At this stage, the embryo has not yet implanted in the uterus. Human...

adult stem cells

Adult Stem Cells in the Small Intestine

Adult stem cells help maintain tissues by replacing old or damaged cells. These undifferentiated cells divide to make more stem cells or progenitor cells, which then become mature, specialized cells. Small groups of adult stem cells are found in many tissues, including the bone marrow, brain, skin, and gut. The small intestine shows this process clearly. Its lining, called the epithelium, is renewed continuously by adult stem cells. It is also the fastest replaced tissue in the human body,...

induced pluripotent stem cells

Reprogramming Mature Cells into Stem Cells

Reprogramming mature cells into stem cells lets scientists turn specialized cells back into an unspecialized state. These cells are called induced pluripotent stem cells, or iPSCs. Like embryonic stem cells, iPSCs are pluripotent, which means they can form all cell types. To make iPSCs, researchers start with mature cells such as skin fibroblasts or blood cells. The cells are grown in culture, and genes for several transcription factors are delivered with a viral vector. The cells then make...

reproductive cloning

Cloning Animals with SCNT

Reproductive cloning can produce a genetically identical copy of an entire organism. In adult animals, this is usually done with somatic cell nuclear transfer, or SCNT. Another way clones can form is by splitting an early embryo, which is similar to how identical twins arise naturally. In SCNT, an egg cell is taken from an animal and its nucleus is removed. This creates an enucleated egg. A somatic cell, which is any body cell that is not a sex cell, is then taken from the animal to be cloned.

genomics

Genomics and DNA Sequencing

Genomics studies the full set of genetic material in an organism. In humans, that genome includes the DNA carried in 23 pairs of chromosomes in the nucleus, along with mitochondrial DNA. Genomics looks at both coding DNA and non-coding DNA, which helps scientists study how living things are built and how they differ. This field is useful in many areas of biology and medicine. Scientists use genomics to build phylogenetic trees, improve crop productivity and sustainability, help with criminal...

key terms and concepts

Learn how antibiotic resistance genes help scientists select transformed bacteria, and why resistance and superbugs are a major health concern.

Antibiotic Resistance in Cloning | JoVE K12

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Antibiotic Resistance in Cloning

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