Exam 1 Prep PDF

Lecture 1 - Hypothesis vs. prediction Hypothesis: usually written in a testable form + "If fertilizer is added to the soil, then the growth rate of the plants will increase.” Prediction: details what you are expecte...

Lecture 1 - Hypothesis vs. prediction Hypothesis: usually written in a testable form + "If fertilizer is added to the soil, then the growth rate of the plants will increase.” Prediction: details what you are expected to see IF HYPOTHESIS IS TRUE + "Plants given 10 grams of fertilizer will grow 2 cm taller in two weeks compared to plants without fertilizer." - Positive vs. negative control Positive: group in an experiment that is known to produce a desired or expected effect. + In an enzyme activity experiment, using a substrate known to react with the enzyme would be a positive control to confirm the enzyme is active. Negative: the group that does not receive the treatment or has conditions where no effect is expected. + In a drug efficacy test, using a placebo group (no active drug) would be a negative control, showing the baseline without treatment. Lecture 2+3 - What is enzyme Catalyst (booster?): a chemical that speeds up a reaction without being consumed in the reaction The enzyme is a catalyst- It is a protein that accelerates the reaction *** all enzymes = catalysts but not all catalysts = enzyme! - How does an enzyme lower the activation energy barrier? What is the exergonic reaction? Exergonic reaction: A chemical reaction that releases energy, it is spontaneous (negative change in free energy) Enzymes function by lowering this activation energy barrier. - How? + By binding to the reactants (substrates) and stabilizing the transition state.  + By orienting the substrates correctly + Providing a favorable environment for the catalytic cycle to occur - Factors that influence catalysis Find time, Processing time + Find time: time for an enzyme to bind to the next substrate + Processing time: enzyme converting substrates into products *** High concentration of substrate decrease find time, but processing time remains the same! What affects reaction rate? + Varying enzyme concentration - The higher the enzyme, the reaction rate increases. However, the reaction rate would eventually level off because the substrate is limiting.  + + Varying substrate concentration - The higher the substrate concentration, the reaction rate will also increase. But, It would eventually level off. Why? 1) Enzyme is saturated and limiting. + Temperature, pH + Efficiency of enzyme + Factors that affect enzyme shape - What is Vmax? V max = maximum rate of reaction (when all active sites are full and processing!) Km = concentration of substrates when the reaction is half maximal. + It is an affinity of enzyme for substrate + HIGHER VMAX + LOWER KM = BETTER (because it means u need less substrate to reach the half V max)  + - What is the role of cofactors? Cofactors are non-protein chemical compounds or metallic ions that assist enzymes in catalyzing biochemical reactions. + essential for the proper functioning of many enzymes, helping them achieve their full catalytic activity. - Competitive vs. noncompetitive inhibitors Competitive inhibitors Noncompetitive inhibitors - Binds to the active site and competes - Binds to another part of the enzyme with substrates and changes the conformation of the - Vmax remains the same since the enzyme function of enzyme is intact - Vmax is lowered due to the conformation change of the enzyme. - How can you design an experiment to tell if a substance is a competitive or noncompetitive inhibitor? Adding competitive or noncompetitive inhibitors and comparing the influence on the Vmax- If Vax is lowered, It would be a non-competitive inhibitor, and If Vmax stays the same that would be a competitive inhibitor,  - How to calculate percent inhibition and what the results mean Percent inhibition varies with different substrate concentrations + competitive inhibitors. But, it is constant for all substrate concentrations + non-competitive inhibitors - What is science communication and what are some barriers to science communication? Science communication = the use of appropriate skills, media, and activities to produce… Awareness, enjoyment, interest, opinions, and understanding Barriers include…scientific literacy, cultural and language differences, misinformation, and lack of trust in scientific sources! Enzyme lab - What reaction does polyphenol oxidase catalyze? - It catalyzes the oxidation of catechol into orthoquinone which produces dark-colored products. - How did we measure enzyme function? We used the spectrophotometer to monitor the change in absorbance over time. The catalysis of polyphenol oxidase and catechol produces a dark-color product. Therefore, by measuring the absorbance value was a way to keep track of the enzyme activity and its reaction. - How do we measure reaction rate? Change in absorbance/ time >> We actually used the 0-2 minutes’ absorbance data to determine the reaction time (slope) - How does substrate affect the reaction rate? As substrate concentration increases, the reaction rate initially increases (since finding time is reduced). But it eventually levels off because the enzyme is saturated and limiting. - How does an enzyme affect the reaction rate? Higher concentrations of the enzyme cause the initial increase of the catalysis reaction rate. However, it also eventually levels off as substrate is limiting. - [product vs. time graphs] and [initiate rate graphs]  - - What is the possible biological role of polyphenol oxidase? Plant defense mechanism- quinones can be toxic to some insects and microbes which protects the plant from being attacked. - Lecture 6 (lab parts IV-IX) review IV: Temperature control- Test the effects of running the reaction at 0 degrees, 20 degrees(room temperature) 45 degrees, and 65 degrees on the rate of enzyme action. (use of ice/warm/hot water baths) + Anticipated reaction rates 0 degrees Slow 20 degrees (room temp) Control 45 degrees Fastest! 65 degrees Slowest! (if the temperature is too high, the enzyme starts to denature. We might see no reaction or slow reaction.) + Experiential Design Tips ~ Prewarm/Pre-cooling the enzyme and substrate-Why? Because water has a high specific heat (It takes time for it to change temperature) ~ Maintaining the temperature during the experiment  V: Freezing/Thawing vs. Boiling the Enzyme-Test the effects of freezing and thawing the enzyme vs. boiling it and then letting it cool + Anticipated reaction rates: ~ Control: never-frozen, never-boiled enzyme (room temp) Frozen > Thawed Enzyme Boiled > Cooled down Enzyme - The faster reaction since - Slower because the enzyme the enzyme maintains its denatures due to high heat. function VI- Potassium arsenite - Potassium arsenite reacts with the adjacent sulfhydryl (SH) groups in a protein and disrupts disulfide bonds necessary to maintain the tertiary structure of the protein + Experimental design 1) Experiment: Use 100 microliter arsenite in a total reaction volume of 1 to 1.2 ml. With varying substrate concentrations 2) Calculate initial rates: graph the result for all reactions, with and without potassium arsenite at each substrate concentration 3) Calculate the percent inhibitions for each substrate concentration. This will tell you whether the inhibitor is competitive or noncompetitive. + Anticipated Results: Arsenite inhibits little or not at all! Because Potassium arsenite is not an inhibitor - There is no SH group in polyphenol oxidase. VII- Para-hydroxybenzoic acid - PHBA has a chemical structure similar to catechol. Is this an inhibitor? + Experimental design: 1) Use 100 microliters of PHBA in a total reaction volume of 1 to 1.2 ml with varying substrate concentrations 2) Calculate initial rates 3) Calculate the percent inhibitions for each substrate concentration.  + Anticipated Results: PHBA shows partial inhibition. Percent inhibition at high substrate is smaller than inhibition at low substrate concentration. Therefore, it is a competitive inhibitor. VIII: Polyphenoloxidase + metal ion cofactor? - Cofactor: something that is required for the reaction to occur + Experimental design 1) Incubate the enzyme with different chelating agents (compounds with tightly bound metal ions) Chelators will strip the metal ions from the active site of the enzyme 2) PTU reacts with copper and potassium cyanide reacts with both copper and ion + Anticipated Results 1) Cyanide and PTU both decrease the reaction rate 2) Our enzyme has a copper cofactor since the presence of PTU lowered the reaction rate. But we are not sure if Cyanide is the cofactor since it reacts with both copper and ion IX: Increasing salt- ions in salt solutions can destabilize the ionic bonds that hold the enzyme in its proper conformation + Experiment 1) Provided with 33 percent salt solution 2) Have to mix it with catechol, buffer, and enzyme to ensure equal volumes and equal concentrations of everything but salt 3) The enzyme should be pre-treated with a salt solution since it may take some time to denature the enzyme + Anticipated results ~ As salt increases, the reaction rate decreases to a certain point. Salt is not really an inhibitor tho since it denatures the enzyme itself. Microbiome - Define the gut microbiome. Where is it located in the human body?  Gut microbiome: all microbiomes present in the gut environment (large intestine=colon) - How is fiber fermented by the gut microbiome? What are the end products Humans lack the enzymes necessary to break down most types of dietary fiber, so when it reaches the large intestine, specific gut microbes digest it through fermentation The gut microbiome breaks down the complex carbs (fiber) into simpler compounds. During the process, the gut microbiome metabolizes anaerobically and The end product of metabolism is: + End product: Hydrogen, CO2, and CH4 (~20 percent of people have methane-producing microbes) - Explain how byproducts of fermentation in the gut can be detected in the breath (How do they get there?) Gases produced during microbial fermentation of fiber in the colon Gases get diffused into the bloodstream Then they get released from the blood into the lungs Gases are exhaled in breath. - Why is it important to collect our breath samples at the end of our exhale? To ensure the exhaled air include the fermentation byproducts since early breath is from upper respiratory tract. - Why do some people produce more methane gas in their breath Because they have microbiome that creates methane as the byproduct of its metabolism. (Using hydrogen to produce methane) - What is the Bristol stool score and how is it related to the gut microbiome Bristol Stool Scale: classify stool into 7 categories based on its shape+consistency + Type 1(hard lumps) - Type 7 (Watery shit) Stool form is influenced by the gut microbiome’s activity - Higher microbiome, healthier shit! (It influences water/nutrient absorption, and fiber fermentation) - Freezer stocks: Why are these prepared? To preserve bacterial strains for long-term Bacteria will be mixed with cryoprotectant to prevent damage during the freezing+thawing process. - PCR: Polymerase Chain Reaction : Technique used to amplify a specific DNA Region, Such as 16S rRNA gene! What are the three steps of each cycle? 1) Denaturation: the double stranded DNA is heated to around 98 degrees, It separates the double strand into single strands. 2) Annealing: lower the temp to 50-65 degrees, allows the primers to bind to the complementary sequences on the DNA template  3) Extension: The temperature si raised to 72 degrees, and the taq DNA polymerase synthesizes a new DNA strand by adding nucleotide to the primers. >> Each cycle results in a doubling of the targe tDNA region- cause exponential increase in DNA quantity over multiple cycles! What is in a PCR cocktail (master mix)? What is the function of each? 1) DNA Template: The DNA you want to amplify (e.g., from a bacterial colony). 2) Primers: Short sequences of DNA that are complementary to the target region and guide DNA polymerase where to start synthesis. 3) dNTPs (deoxynucleotide triphosphates): Building blocks of DNA (adenine, thymine, cytosine, guanine). 4) Buffer: Maintains the optimal pH and salt conditions for the reaction. 5) MgCl₂: A cofactor required for DNA polymerase activity. 6) Taq DNA Polymerase: The enzyme that synthesizes new DNA strands by adding dNTPs to the primers​ What is the purpose of positive and negative controls in PCR? + Positive control: to make sure the master mix and the thermocycler is functioning correctly. + Negative control: detect contamination in the reagents. - Lab techniques Quantifying bacteria using the standard plate count method Spread plating Isolating bacteria using streak plating Aseptic technique Fasting breath samples and analysis