An antibiotic is an antimicrobial chemical that kills or inhibits the growth of microorganisms. Antibiotics have been used for a long time to cure many deadly infections, such as bubonic plaque, diphtheria, and tuberculosis.
The mechanisms of action of antibiotics depends on their structures and their affinity level with the target in the microorganism cells.
Mechanisms by which antibiotics target bacteria are:
- Inhibiting the synthesis of cell wall
- Inhibiting the function of cell membrane
- Inhibiting the synthesis of protein
- Inhibiting the synthesis of nucleic acid
- Inhibiting other metabolic processes
After the first discovery of an antibiotic, penicillin, in 1928 by Alexander Fleming, many antibiotics have been isolated and used for the treatment of many diseases. Unfortunately, many bacterial strains have developed resistance against widely used antibiotics.
Commonly Use Antibiotics, Their Mode of Action, and Mechanism of Resistance
Ampicillin is an antibiotic with a β-lactam ring that inhibits penicillin-binding proteins (PBPs), involved in peptidoglycan biosynthesis. When the PBPs interacts with β-lactam rings, these proteins can’t catalyze the synthesis of new peptidoglycan, disrupting the formation of the bacterial cell wall.
Bacteria become resistant to ampicillin by producing β-lactamase enzyme. This enzyme cleaves the β-lactam ring of ampicillin to inactivate it. Many cloning vectors with a resistance gene, bla gene, produce β-lactamase enzyme.
Chloramphenicol is a broad-spectrum antibiotic, targeting a wide range of microorganisms. This antibiotic inhibits the activity of ribosomal peptidyl transferase. As a result, it inhibits protein synthesis.
Bacteria develop chloramphenicol resistance by using the activity of chloramphenicol acetyl transferase (CAT) to modify chloramphenicol.
Examples of Different Mechanisms of Antibiotic Resistance. 1) Antibiotic degradation by β-lactamases. 2) Antibiotic modification by chloramphenicol acetyl transferase (CAT) and amino-phospotransferase (APH). 3) Antibiotic efflux by Tet proteins. 4) Ribosome protection by Tet proteins.
Kanamycin is a member of aminoglycoside family of antibiotics. This antibiotic interacts with three ribosomal proteins and rRNA in the 30S ribosomal subunit, causing the inhibition of protein synthesis.
Resistance to kanamycin involves the activity of amino-phosphotransferase enzymes.To inactivate and modify kanamycin, an amino-phosphotransferase transfers a phosphate group from their ATP (Adenine Triphosphate) to kanamycin. Two examples of these enzymes are Aph (3’)-I and Aph (3’)-II.
Tetracyclines bind to a site on the 30S ribosomal subunit to prevent the attachment of aminoacyl tRNA to the acceptor site and inhibit the synthesis of protein.
The resistance against tetracyclines involves Tet proteins. Tet proteins catalyze the antibiotic efflux (by pumping tetracyclines out of the cell) or protect the ribosome (by blocking tetracyclines from binding to the ribosome). Tet(A), Tet(B), Tet(C), Tet(D), Tet(E), Tet(G) and Tet(H) are efflux proteins. Tet(M) and Tet(O) are examples of proteins involved in ribosomal protection.
How Do Bacteria Acquire Antibiotic Resistance?
Bacteria can obtain antibiotic resistance from:
- Gene mutations, for example mutations in genes encoding protein targets of an antibiotic.
- Exchanging of resistance genes using the plasmids or other mobile genetic elements.
The resistance gene can produce:
- enzymes that inactivate the antibiotic.
- proteins to modify the antibiotic target.
- enzymes or proteins to replace the antibiotic target and reduce the binding of the antibiotic to its target.
- proteins to reduce the uptake of the antibiotic inside the cell.
The Mechanisms of Horizontal Gene Transfer by Bacteria to Acquire Resistance
The exchange or horizontal gene transfer (HGT) of resistance genes by bacteria uses three main strategies:
- Transformation: uptake of naked DNA by a competent cell.
- Transduction: transfer of DNA material by a bacteriophage.
- Conjugation: transfer of DNA through cell-to-cell contact using a mating pilus.
The Use of Antibiotic Resistance Genes in Molecular Cloning
How Does Antibiotic Selection Work in Molecular Cloning?
Bacteria that can’t get antibiotic resistance naturally will not survive and grow in the growth medium containing antibiotics. Based on this, molecular biologists design special plasmid vectors.
These vector tools carry a specific DNA insert that the researcher want to amplify and an antibiotic resistance gene as a selectable marker. Competent bacteria cells will take up these plasmid vectors inside the cells through artificial transformation (by heat shock or electroporation).
After artificial transformation and antibiotic selection, only bacteria containing the plasmid vector with a resistance gene survive in the cell culture. During antibiotic selection, antibiotics kill bacteria that don’t have the plasmid vector with the resistance gene in the cell.
Common Antibiotic Selection Problems in Molecular Cloning
Below are common problems during antibiotic selection and a simple troubleshooting guide for these problems:
1. No Colonies Grow on the Plate
- Check if the competent cells is still viable.
- Check if the correct antibiotic is used for selection.
- Check if your antibiotic concentration is higher than the recommended concentration.
2. Too Many Small Colonies Grow on the Plate
- Check if the antibiotic concentration is too low.
- Check if the temperature of the plate is not too hot when adding the antibiotic.
- Use a stirrer to mix the antibiotic evenly in the growth medium.
- Check if the antibiotic stock is not too old. For tips on how to check that, watch our Kirby Bauer Assay video below:
Tips to Avoid Antibiotic Selection Problems
- Check the genotype of competent cells before cloning. Some cloning strains may be naturally resistant to some antibiotics. Below are GoldBio competent cells with antibiotic resistance.
- Make sure you use the correct antibiotic in the growth medium.
- Check the protocol for the recommended antibiotic concentration to use.
DH10B Chemically Competent E. coli Cells (Catalog No. CC-100)
DH10B Electrocompetent E. coli Cells (Catalog No. CC-200)
DH5-alpha Chemically Competent E. coli Cells (Catalog No. CC-101)
BL21 (DE3) Chemically Competent E. coli Cells (Catalog No. CC. 103)
DL39 (DE3) Chemically Competent E. coli Cells (Catalog No. CC-104)
DH10B-Pro™ Electrocompetent E. coli Cells (Catalog No. CC-201)
DH5-alpha Electrocompetent E. coli Cells (Catalog No. CC-203)
BL21 (DE3) Electrocompetent E. coli Cells (Catalog No. CC-204
GV3101 Agrobacterium Electrocompetent Cells (Catalog No. CC-207)
AGL-1 Agrobacterium Electrocompetent Cells (Catalog No. CC-208)
Popular Antibiotics Products
Ampicillin (Sodium), USP Grade (Catalog No. A-301)
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Tetracycline Hydrochloride (Catalog No. T-101)
Carbenicillin (Disodium), USP Grade (Catalog No. C-103)
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