Antibiotic Resistance and Protein Synthesis

• Antibiotic resistance poses a major public health crisis due to increasing instances of bacteria surviving antibiotic treatments.
• Over one million deaths occur annually from antibiotic-resistant bacterial infections, underscoring the urgency of the issue.
• Antibiotics are vital for treating bacterial infections by targeting bacterial functions without harming human cells.
• Antibiotics disrupt cellular processes, especially translation, essential for protein synthesis.

Mechanisms of Antibiotic Resistance

• Bacteria resist antibiotics by evading their effects through mechanisms that target specific antibiotics or drug classes.
• Resistance mechanisms include chemical modification of antibiotics and active efflux, where bacteria pump out the antibiotic.
• Antibiotic resistance genes are DNA segments encoding proteins that enable bacteria to survive antibiotic exposure.

Understanding Protein Synthesis

• Translation, the process by which ribosomes synthesize proteins using mRNA, is crucial for cellular function.
• Antibiotics disrupting translation prevent bacteria from producing essential proteins, leading to cell death.
• BioBits is a cell-free protein synthesis system mimicking bacterial protein synthesis in vitro, allowing observation of antibiotic effects without live bacterial cultures.
• The system employs DNA instructions to produce proteins, like the red fluorescent protein (RFP), indicating successful translation.

Experimental Design and Objectives

• Experiments aim to characterize an antibiotic resistance gene and two antibiotics to understand their mechanisms of action.
• These experiments also help confirm antibiotics block translation and identify which antibiotic is affected by the resistance gene.
• Red fluorescence of RFP serves as a visual indicator of successful protein synthesis in the presence of antibiotics.
• Susceptible bacteria will stop dividing and die with antibiotics, while resistant bacteria will thrive.
• These experiments provide insights into studying antibiotic resistance without traditional culturing methods, enhancing understanding of bacterial survival.

Overview of Protein Synthesis and Antibiotics

• Protein synthesis involves transcription and translation, where DNA is converted into proteins.
• Ribosomes are important in translation, essential for facilitating the assembly of amino acids into proteins.
• Disruption of protein synthesis can occur from antibiotics, targetting stages of protein synthesis.
• The red fluorescent protein (RFP) indicates that translation has occurred successfully.
• Kanamycin and streptomycin inhibit protein synthesis by binding to bacterial ribosomes and preventing translation.
• These antibiotics are aminoglycosides and are effective against bacteria.
• The mechanism of action involves interference with the ribosomal function, leading to cell death.
• Antibiotic resistance genes, such as those encoding aminoglycoside kinases, can provide bacteria with the ability to survive aminoglycosides.
• Resistance mechanisms involve the modification of the antibiotic or target site, rendering the antibiotic ineffective.

Experimental Design and Predictions

• Experiments aim to test whether kinase DNA can confer resistance to kanamycin or streptomycin.
• The presence of kinase DNA is expected to allow continued translation in the presence of antibiotics, indicated by RFP fluorescence.
• Prediction outcomes are based on the presence of Biobits, kinase DNA, antibiotics, and RFP DNA.
• The predictions are based on how antibiotics interact with ribosomes and the role of resistance genes.
• Fluorescence observations after the experiment will help confirm or refute the predictions made.
• The results will provide insights into the effectiveness of the kinase gene in conferring antibiotic resistance.

Mechanisms of Antibiotic Resistance

• Antibiotic resistance occurs when bacteria evolve mechanisms to resist the effects of drugs that once killed them or inhibited their growth.
• Resistance genes often encode proteins that can modify or degrade the antibiotic, preventing it from exerting its effects.
• Horizontal gene transfer can spread resistance genes among bacterial populations, complicating treatment options.
• Antibiotic resistance genes pose a challenge in clinical settings, leading to treatment failures.
• Resistance can be combatted through the development of antibiotics and combination therapies.
• Monitoring and controlling antibiotic use is essential to slow the spread of resistance.

Targeting Bacterial Structures

• Antibiotics often target unique bacterial structures, such as the cell wall, which are not present in human cells.
• Disrupting cell wall synthesis leads to bacterial lysis and death, making it an effective target for antibiotics.
• Selectivityis crucial for minimizing toxicity to human cells while effectively treating bacterial infections.
• Differences in ribosomal structure between bacteria and eukaryotes allow for selective targeting of bacterial ribosomes by certain antibiotics.
• Understanding these structural differences is key to developing antibiotics that are effective against bacteria without harming human cells.

Experimental Observations and Data Analysis

• Data from the experiment includes the presence or absence of fluorescence in each tube at specified time intervals.
• Observations will be recorded for both kanamycin and streptomycin treatments, noting any changes in fluorescence over time.
• The color of fluorescence will also be documented, providing additional information on the success of translation.
• Analyzing the data will help determine the effectiveness of the kinase gene in conferring resistance to the antibiotics used.
• The results will be compared against predictions made prior to the experiment to assess the accuracy of the hypotheses.
• Understanding the outcomes is important for the broader knowledge of antibiotic resistance mechanisms.