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

K12 Standards

Louisiana Student Standards for Science

High School

Life Sciences

from-molecules-to-organisms-structures-and-processes

From Molecules To Organisms: Structures And Processes

HS-LS1-8

Antibiotic Selection

- [Istruttore] La selezione antibiotica

viene in genere utilizzata

per garantire che una colonia batterica

contenga un plasmide con
alcune caratteristiche chiave.

Un gene di resistenza agli antibiotici

e un gene di interesse che un
ricercatore vuole esprimere.

Perch&#xE9; i batteri assorbano il plasmide,

entrambi devono essere incubati insieme

e quindi subire uno shock termico

per promuovere l&#x27;incorporazione.

Non tutti i batteri
assorbiranno con successo

una copia del plasmide.

Poich&#xE9; l&#x27;obiettivo

&#xE8; isolare i batteri che
possiedono una copia,

i ricercatori aggiungono
parte della coltura batterica

su una piastra contenente un antibiotico.

Solo quei batteri che
hanno assunto il plasmide

che contiene il gene della resistenza

saranno in grado di
degradare l&#x27;antibiotico

e crescere sulla piastra.

Nei prossimi giorni un singolo batterio

former&#xE0; colonie che potranno

essere selezionate per ulteriori lavori.

Courvalin, P. 2016. "Perché la resistenza agli antibiotici è una malattia emergente mortale?" Microbiologia clinica e infezione 22 (5): 405–7. [<a href="https://doi.org/10.1016/j.cmi.2016.01.012" target="__blank">Sorgente</a>]

Daubin, Vincent e Gergely J. Szöllősi. 2016. "Trasferimento genico orizzontale e storia della vita". Prospettive di Cold Spring Harbor in biologia 8 (4). [<a href="https://doi.org/10.1101/cshperspect.a018036" target="__blank">Sorgente</a>]

Hiltunen, Teppo, Marko Virta e Anna-Liisa Laine. 2017. "Resistenza agli antibiotici in natura: una prospettiva eco-evolutiva". Transazioni filosofiche della Royal Society B: Scienze biologiche 372 (1712). [<a href="https://doi.org/10.1098/rstb.2016.0039" target="__blank">Sorgente</a>]

selezione antibiotica

<h4>Panoramica</h4>

I ricercatori utilizzano geni di resistenza agli antibiotici per identificare i batteri che possiedono un plasmide contenente il loro gene di interesse. La resistenza agli antibiotici si verifica naturalmente quando una mutazione spontanea del DNA crea cambiamenti nei geni batterici che eliminano l&#x27;attivit&#xE0; antibiotica. I batteri possono condividere questi nuovi geni della resistenza con la loro prole e altri batteri. L&#x27;abuso e l&#x27;abuso di antibiotici hanno creato una crisi di salute pubblica, poich&#xE9; i batteri resistenti e multiresistenti continuano a svilupparsi.

<h4>La resistenza agli antibiotici &#xE8; uno strumento essenziale per l&#x27;ingegneria genetica</h4>

Gli antibiotici, come la penicillina, sono farmaci che uccidono o fermano la crescita batterica. I batteri che hanno acquisito naturalmente o artificialmente i geni della resistenza agli antibiotici non rispondono agli antibiotici. Gli scienziati lo sfruttano progettando plasmidi, piccoli pezzi di DNA auto-replicanti, che portano sia un gene di resistenza agli antibiotici che un gene di interesse. La resistenza agli antibiotici &#xE8; parte integrante della clonazione del DNA che consente a un ricercatore di identificare le cellule che hanno assorbito un DNA di interesse.

Il DNA di interesse del ricercatore viene introdotto nelle cellule batteriche utilizzando un processo chiamato trasformazione. La trasformazione batterica comporta la creazione temporanea di piccoli fori nella parete cellulare batterica per consentire l&#x27;assorbimento di DNA esterno come un plasmide. Solo alcune cellule batteriche assorbono nuovo DNA. Poich&#xE9; il plasmide comprende sia il DNA di interesse che un gene che conferisce resistenza a un antibiotico specifico, l&#x27;applicazione dell&#x27;antibiotico alle cellule batteriche (cio&#xE8; la selezione degli antibiotici) pu&#xF2; aiutare a determinare quali cellule sono state geneticamente modificate.

Il ricercatore diffonde le cellule batteriche su una piastra di coltura contenente un antibiotico scelto. Solo i batteri contenenti il gene della resistenza agli antibiotici sopravvivono e crescono sulla piastra. Dopo alcuni giorni, il ricercatore pu&#xF2; selezionare una colonia batterica da sperimentare per ulteriori esperimenti, come gli studi sull&#x27;espressione genica. Dopo la selezione degli antibiotici, il ricercatore tester&#xE0; ulteriormente i batteri utilizzando altri metodi (ad esempio, PCR) per confermare che il DNA di interesse &#xE8; corretto. Spesso si verificano errori come il plasmide che non contiene affatto il gene di interesse.

<h4>I batteri possono acquisire resistenza agli antibiotici naturalmente</h4>

I batteri possono acquisire resistenza agli antibiotici attraverso mutazioni spontanee del DNA che alterano le proteine prodotte dalla cellula. I batteri resistenti possono produrre proteine che causano il degrado dell&#x27;antibiotico, pompato fuori dalla cellula o impedito di interagire con il suo bersaglio. Ad esempio, la vancomicina antibiotica inibisce la sintesi della parete cellulare batterica. Alcuni batteri hanno sviluppato resistenza a questo antibiotico modificando i tipi di sottounit&#xE0; proteiche, gli amminoacidi, utilizzati nell&#x27;assemblaggio della parete cellulare in quelli che non sono influenzati dalla vancomicina.

Una volta che i geni della resistenza agli antibiotici emergono, i batteri possono trasmetterli alla loro prole. I batteri possono anche acquisire geni di resistenza agli antibiotici da altri batteri della stessa specie o di specie diverse attraverso un processo chiamato trasferimento genico orizzontale (HGT). Ci sono tre meccanismi di HGT: trasformazione, coniugazione e trasduzione. I geni di resistenza agli antibiotici si trovano spesso in plasmidi o trasposoni, pezzi di DNA che si trasferiscono facilmente tra batteri, che vengono scambiati durante l&#x27;HGT. Di conseguenza, nuovi tipi di resistenza agli antibiotici possono diffondersi rapidamente a pi&#xF9; tipi di batteri infettivi.

<h4>L&#x27;uso eccessivo clinico e l&#x27;uso improprio di antibiotici producono &#x22;Superbatteri&#x22;</h4>

Gli antibiotici sono un trattamento critico per le infezioni batteriche. Tuttavia, il loro uso pu&#xF2; causare batteri a diventare resistente e rendere l&#x27;antibiotico inefficace, che porta a infezioni non curabili e potenzialmente mortali. L&#x27;abuso di antibiotici e l&#x27;abuso, ad esempio l&#x27;uso di antibiotici per trattare infezioni virali (piuttosto che batteriche) o per aumentare la crescita del bestiame, &#xE8; problematico perch&#xE9; favorisce la resistenza.

Gli antibiotici causano resistenza per evolvere perch&#xE9; uccidono i batteri sensibili e lasciano solo gli individui resistenti. I batteri sopravvissuti si dividono rapidamente, producendo prole con la stessa resistenza agli antibiotici. Quando gli antibiotici sono abusati, questa pressione di selezione provoca il numero di batteri resistenti nella popolazione a salire rapidamente. Questo &#xE8; un grande problema di salute pubblica perch&#xE9; aumenta la resistenza agli antibiotici e crea &#x22;superbatteri&#x22; che sono resistenti a antibiotici multipli. L&#x27;uso eccessivo e l&#x27;abuso di antibiotici possono eventualmente esaurire le opzioni di trattamento per le infezioni batteriche.

Selezione antibiotica

Overview

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

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KEY TERMS AND CONCEPTS

TERMINI E CONCETTI CHIAVE

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

DNA extraction is the process of removing and purifying DNA from cells. It is used in many biotechnology and research applications, including molecular cloning. The main goal is to free DNA from the cell and prepare it for later study or use. The first step is cell lysis, which means breaking cells open. Researchers may use a homogenizer, sonicator, or bead beater to physically lyse the cells. They also often add detergent to chemically disrupt the lipid-based cell membranes. This helps...

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

PCR is a laboratory method for making many copies of a DNA segment. It can turn a small amount of DNA into millions or even billions of copies in just a few hours. The process uses a heat-resistant DNA polymerase inside a machine called a thermocycler. PCR was developed by Kary Mullis in 1983, and he received the 1993 Nobel Prize in Chemistry for it. The method is fast, relatively inexpensive, and precise. It has become useful for molecular cloning, gene mutagenesis, pathogen detection, gene...

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 selection

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

Knockout mice help researchers study gene function by showing what happens when a gene is turned off. These engineered animals are also useful for modeling human diseases such as cancer, Parkinson’s disease, and diabetes. A specific gene can be targeted by using a targeting vector, which is a piece of engineered DNA that replaces the normal gene and inactivates it. The vector has matching DNA sequences on both ends. These homologous sequences let the vector swap in for the target gene through...

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

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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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Nucleic Acids and Nucleotides

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Types of RNA

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Antibiotic Resistance in Cloning

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