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

- [Instructeur] Antibiotica-selectie wordt meestal gebruikt om ervoor te zorgen dat een bacteriekolonie een plasmide bevat met een aantal belangrijke kenmerken: een antibioticumresistentiegen en een specifiek gen waarop een onderzoeker genexpressie wil toepassen.
 Opdat de bacteri&#xEB;n de plasmide zouden opnemen, moeten beide samen ge&#xEF;ncubeerd worden en vervolgens een heat shock ondergaan om de incorporatie te bevorderen.
 Niet elke bacterie zal met succes een kopie van de plasmide opnemen.
Omdat het doel is om bacteri&#xEB;n die een kopie bezitten, te isoleren, voegen onderzoekers wat van de bacteriecultuur toe op een schaaltje met een antibioticum.
 Enkel die bacteri&#xEB;n die de plasmide hebben opgenomen, dat het resistentiegen bevat, zullen het antibioticum kunnen afbreken en groeien op het schaaltje.
 In de loop van de volgende dagen, zullen afzonderlijke bacteri&#xEB;n kolonies vormen, die dan geselecteerd kunnen worden voor verder werk.

selectie van antibiotica

<h4>Overzicht</h4>

Onderzoekers gebruiken antibioticaresistentiegenen om bacteri&#xEB;n te identificeren die een plasmide bezitten dat het gen van interesse bevat. Antibioticaresistentie komt van nature voor wanneer een spontane DNA-mutatie veranderingen in bacteri&#xEB;le genen veroorzaakt die de antibiotische activiteit elimineren. Bacteri&#xEB;n kunnen deze nieuwe resistentiegenen delen met hun nakomelingen en andere bacteri&#xEB;n. Het overmatig gebruik en misbruik van antibiotica heeft een volksgezondheidscrisis veroorzaakt, omdat resistente en multiresistente bacteri&#xEB;n zich blijven ontwikkelen.

<h4>Antibioticaresistentie is een essentieel instrument in de genetische manipulatie</h4>

Antibiotica, zoals penicilline, zijn medicijnen die de groei van bacteri&#xEB;n doden of stoppen. Bacteri&#xEB;n die op natuurlijke of kunstmatige wijze antibioticaresistentiegenen hebben verworven, reageren niet op antibiotica. Wetenschappers maken hier gebruik van door plasmiden te ontwerpen &#x2013; kleine, zelfreplicerende stukjes DNA &#x2013; die zowel een gen voor antibioticaresistentie als een interessant gen bevatten. Antibioticaresistentie is een integraal onderdeel van DNA-klonering waarmee een onderzoeker cellen kan identificeren die een interessant DNA hebben geabsorbeerd.

Het DNA van de onderzoeker wordt in bacteri&#xEB;le cellen ge&#xEF;ntroduceerd met behulp van een proces dat transformatie wordt genoemd. Bacteri&#xEB;le transformatie omvat het tijdelijk cre&#xEB;ren van kleine gaatjes in de bacteri&#xEB;le celwand om de opname van extern DNA, zoals een plasmide, mogelijk te maken. Slechts enkele bacteri&#xEB;le cellen nemen nieuw DNA op. Omdat het plasmide zowel het DNA van belang bevat als een gen dat resistentie tegen een specifiek antibioticum verleent, kan het toepassen van het antibioticum op de bacteri&#xEB;le cellen (d.w.z. antibioticaselectie) helpen bepalen welke cellen genetisch gemodificeerd zijn.

De onderzoeker spreidt de bacteriecellen uit op een kweekplaat met daarin een gekozen antibioticum. Alleen bacteri&#xEB;n die het antibioticaresistentiegen bevatten, overleven en groeien op de plaat. Na een paar dagen kan de onderzoeker een bacteriekolonie selecteren om te kweken voor verdere experimenten&#x2013;zoals onderzoek naar genexpressie. Na de antibioticaselectie zal de onderzoeker de bacteri&#xEB;n verder testen met behulp van andere methoden (bijvoorbeeld PCR) om te bevestigen dat het DNA van interesse correct is. Er komen vaak fouten voor, zoals dat het plasmide het gen van interesse helemaal niet bevat.

<h4>Bacteri&#xEB;n kunnen op natuurlijke wijze antibioticaresistentie verwerven</h4>

Bacteri&#xEB;n kunnen antibioticaresistentie verwerven door spontane DNA-mutaties die de door de cel geproduceerde eiwitten veranderen. Resistente bacteri&#xEB;n kunnen eiwitten produceren die ervoor zorgen dat het antibioticum wordt afgebroken, uit de cel wordt gepompt of wordt verhinderd om in wisselwerking te treden met het doelwit. Het antibioticum vancomycine remt bijvoorbeeld de synthese van de bacteri&#xEB;le celwand. Sommige bacteri&#xEB;n hebben resistentie tegen dit antibioticum ontwikkeld door de typen eiwitsubeenheden &#x2013;aminozuren&#x2013; die worden gebruikt bij de assemblage van hun celwand te veranderen in typen die niet worden be&#xEF;nvloed door vancomycine.

Zodra antibioticaresistentiegenen ontstaan, kunnen bacteri&#xEB;n deze doorgeven aan hun nakomelingen. Bacteri&#xEB;n kunnen ook antibioticaresistentiegenen verwerven van andere bacteri&#xEB;n van dezelfde of een andere soort via een proces dat horizontale genoverdracht (HGT) wordt genoemd. Er zijn drie mechanismen van HGT: transformatie, conjugatie en transductie. Antibioticaresistentiegenen worden vaak aangetroffen in plasmiden of transposons &#x2013; stukjes DNA die gemakkelijk worden overgedragen tussen bacteri&#xEB;n &#x2013; die worden uitgewisseld tijdens HGT. Als gevolg hiervan kunnen nieuwe soorten antibioticaresistentie zich snel verspreiden naar meerdere soorten infectieuze bacteri&#xEB;n.

<h4>Klinisch overmatig gebruik en misbruik van antibiotica leidt tot &#x2018;superbacteri&#xEB;n&#x2019;</h4>

Antibiotica zijn een cruciale behandeling voor bacteri&#xEB;le infecties. Het gebruik ervan kan er echter voor zorgen dat bacteri&#xEB;n resistent worden en het antibioticum ineffectief maken, wat kan leiden tot onbehandelbare en mogelijk dodelijke infecties. Overmatig gebruik en misbruik van antibiotica &#x2013; bijvoorbeeld het gebruik van antibiotica om virale (in plaats van bacteri&#xEB;le) infecties te behandelen of om de groei van vee te vergroten &#x2013; is problematisch omdat het de resistentie bevordert.

Antibiotica zorgen ervoor dat resistentie evolueert omdat ze gevoelige bacteri&#xEB;n doden en alleen de resistente individuen achterlaten. De overlevende bacteri&#xEB;n delen zich snel en produceren nakomelingen met dezelfde antibioticaresistentie. Bij overmatig gebruik van antibiotica zorgt deze selectiedruk ervoor dat het aantal resistente bacteri&#xEB;n in de bevolking snel stijgt. Dit is een groot probleem voor de volksgezondheid omdat het de resistentie tegen antibiotica vergroot en &#x2018;superbacteri&#xEB;n&#x2019; cre&#xEB;ert. die resistent zijn tegen meerdere antibiotica. Voortdurend overmatig gebruik en misbruik van antibiotica kan uiteindelijk de behandelingsmogelijkheden voor bacteri&#xEB;le infecties uitputten.

Selectie van 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

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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

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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.

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adult stem cells

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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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