Bio Lab Midterm

Which of these proteins would you propose normally act to promote cell division? Which proteins mights normally act to inhibit cell division?

Actin (large values) would normally act to promote cell division and BCl-2 (small values) would inhibit.

hypotonic solution

GREATER CONCENTRATION INSIDE CELL THAN OUTSIDE, MORE WATER ON OUTSIDE THEREFORE MOVE IN external medium is more dilute, hence water enters with its concentration gradient. under extreme conditions the cell will swell and lyse

hypertonic solution

GREATER CONCENTRATION OUTSIDE CELL THAN INSIDE, MORE WATER INSIDE THEREFORE WANTS TO MOVE OUT external medium is more concentrated, hence water leaves the cell. this results in shrinkage called "crenation" or "plasmolysis"

which of the following would be expected to decrease the distance molecules migrate in an electrophoresis experiment? a) increasing the concentration of agarose in the gel b) increasing the time the experiment runs c) increasing the voltage applied d) none of the above e) all of the above

a) increasing the concentration of agarose in the gel

leukemia cells vs. normal blood

leukemia smear, there are more white blood cells present than a normal blood smear. the leukemia smear also had RBC clumped together, while the normal blood smear has distinct circular RBC and WBC. White blood cells are circular shaped in the leukemia smear as well, just the quantities are different.

if a red onion cell is placed in a hypotonic solution it will

lose water

% cells transformed

measure of success of transformation % = number of transformed colonies on plate #1/number of bacteria originally added to plate #1 Transformation efficiency = 34 transformants/2,000 cells plated x 100% = 1.7% transformants/cells plated

molar concentration of sucrose in 30% (g/100mL) solution?

molar conc (mol/L) = 30. g/100 mL • 1000 mL/1L • 1 mole/342 g = 0.877 M

Using the frequencies in Table, calculate the probability of finding another person with this exact combination. to suspect #2's

multiply all the genotype frequencies together

suppose a forensic scientist had only analyzed FGA and D55818. what percent of the population has the same combination as suspect #2?

multiply frequencies for each locus to each other and then by 100 to get a percent

What is orientation and why is it required for successful transformation?

ori is the origin of the pGLO plasmid where replication occurs. Required because makes sure whole plasmid gets replicated and put into all the cells

lab 2 analysis

osmotic pressure of 20% sucrose solution: 200. g/L • 1 mol/342 g sucrose = 0.585 πosm = (0.585 mol/L)(R)(295K) = 14.2 atm cumulative change (∆) in weight of each bag versus time (LINE GRAPH) graph of rate (mg/min/cm^2) vs. concentration (%) - calculated slope (mg/min.) for each curve on Figure 1 and divided by the surface area of corresponding bag (cm^2) (BAR GRAPH)

If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located?

plates 1 & 2 because transformation occurred

peptide backbone

stabilized by folding and twisting into a complex 3-D configuration

lab 3

studied: 1. the activity of lactose enzyme 2. the effect of enzyme concentration on activity 3. the effect of temperature on enzyme activity

What would you expect the gel to look like if the electrophoresis had been allowed to continue overnight?

the Gel would be empty cause bands travel off of gel

osmotic pressure of a solution is proportional to

the concentration of solution, gas constant, and temperature

study I: effects on DNA replication (lab 1)

the control developed most over time while subiolsamide was less radioactive and cladribine was most effective (LINE GRAPH)

isotonic solutions

there is no net gainer loss of water because osmolarity of the cell and its environment are equal, no osmotic gradient

which of the following is true of enzymes?

they accelerate and steer chemical processes in organisms

t/f: after bacteria are spread on agar plates each cell will multiply to form a colony, which appears as a dome of cells on the agar surface

true

t/f: bacteria produce small circular molecules of DNA called plasmids

true

t/f: bacteria that have the pGLO plasmid will survive in the presence of the antibiotic ampicillin

true

t/f: in lab 7 you will transform bacteria with a gene taken from jellyfish

true

Pie charts

Used to summarize a set of data that is linked together, and each piece of data is part of a whole. For example, the world's population can be divided into pie slices that each represents a country's population.

Bar graphs

Used when the independent variables are discrete: they are unique and unshared, and no values fall between them. For example, if you wish to illustrate the relationship between average life span (dependent variable) and insect species (independent variable), use a bar graph.

how will the formation of producers be detected in today's lab?

colorimetric measurement using a spectrophotometer

what is the natural role of lactase in mammals?

hydrolysis of a disaccharide

t/f: the transfer of the human insulin gene into bacteria is called "genetic transformation"

true

active site

a region on an enzyme that binds to a protein or other substance during a reaction

which molecule is expected to have the fastest migration rate and which is expected to have the slowest? a) 4/5 b) 3/1 c) 3/5 d) 2/5 e) none of the above

b) 3/1

if you were to graph the amount of WBCs in the normal vs. leukemia smear, what type of graph would you use? x/y axis?

bar graph, x = normal smear and leukemia smear, y = number of white blood cells in each smear

study II: effects on gene expression (lab 1)

the control (no drugs) and subiolsamine are very similar in amount of protein except for CD2 and CD63. actin is the same for all three groups. in conclusion, depending on the protein the affect of the drug varies (BAR GRAPH)

osmotic pressure (π)

πosm = CRT C = total solute concentration in moles/liter R = gas constant = 0.082 L•atm/˚K*mol T = temperature Kelvin = ˚K = [273 + ˚C] proportional to the concentration gradient

concentration

a way of stating the amount of that molecule dissolved in a certain volume of solution [ g/L]

gel electrophoresis is useful for a) all of the above b) providing evidence in criminal cases c) determining paternity d) diagnosing genetic diseases

a) all of the above

why was it important to compare all samples to "blank tube"

able to compare how much effect, if any, a component has towards the result. its used to calibrate the spectrometer, and has all the same materials inside but no reaction occurs because Na2CO3 added in beginning therefore no color

enzyme reactions

action of enzyme depends on the shape of active site matching shape of substrate

gel electrophoresis is a powerful and widely used analytical method that separates molecules on the basis of their a) size b) shape c) all of the above d) charge

c) all of the above

standard curve

can be used to estimate the concentration of the enzyme solution use data from a sample tube that was treated to the same conditions as those used to create the standard curve

cell culture studies

cancerous cells can be removed from a person and grown in artificial media

Line graphs

May be used to compare changes over short and long periods of time. Line graphs are best when the independent variables are in a continuous series, such as time or substrate concentration, which have an unlimited number of values between points. Line graphs plot the averages of the dependent variable collected at specific points along the independent variable, and typically a line connects the points.

folds

2:4 ratio, 2mL solution, 4mL water 1:2 ratio = 3 fold add ratio = fold

rate

rise/run kept increasing

based on observations, what type of symptoms do you think occur with leukemia and why?

symptoms could be difficulty breathing, lightheadedness or dizziness because RBC carry oxygen. leukemia smears red blood cells are clumped together and deformed causes a lack of circulation for oxygen to get to the body causing these symptoms.

what does it mean to say that a plant cell is "isotonic" to its surrounding solution?

the cytosol has the same osmolarity as the surrounding fluids

lactase

the enzyme responsible for catalyzing the digestion of lactose into galactose and glucose functions in intestinal tract of humans and other mammals

Create your graph

1. Determine the appropriate range for each axis 2. Plot your data accurately (For line graphs, connect points. Use standard error bars if indicated. If you do not have data for the zero point, do not extrapolate to the zero point) 3. Label each axis (name and units) 4. If more than one data set is plotted, provide a legend (right or graph) that states the difference between the two (or more) lines 5. Include a caption, placed below the graph (short description of the experimental treatment groups, sample sizes, and pattern; if error bars, describe whether depicting standard error or standard deviation)

instructions for using the compound microscope

1. clean the objective and ocular lenses with lens cleaning solution and lens paper if needed. DON'T USE A KIMWIPE 2. clean the microscope slide with a clean tissue, if needed 3. always begin on the lowest power objective lens (4X). initially focus with the coarse control knob, and then use the fine focus knob to acquire a crisper image 4. once you find and focus on the cells of 4X, you can go up in magnification to 10X, then 40X if needed, adjusting the fine focus as you go (don't use the coarse focus here; you will lose the specimen!) the 100X objective requires the use of oil; as you instructor before using 5. you may want to adjust the diaphragm; closing the diaphragm to let in less light (smallest aperture) may let you see the cells more clearly 6. after you are done looking at the slide, rotate the high power objective lenses out of the way before removing a slide in order not to scrape them

Estimating cell length

1. estimate the fraction of the field width occupied by sample 2. in table, find row with the objective lens that you are using to view your sample 3. multiply that fraction by the field diameter of that objective to find the sample's size ex. (1/10)(450) = 45

how would observations change if beaker contained 20% sucrose instead of water?

10% concentration would show an inverse relationship, sloping negatively because the tube would lose mass 20% would show an horizontal line due to the solution inside tube being isotonic with surroundings 30% would still be positive, but slope would be less than it is in water

each enzyme has a characteristic temp at which it operates most efficiently (optimal temp) what is the optimal temp for lactase? make sense?

45˚C makes sense because it is not extreme, about median temperature for lactase enzyme to thrive in and maintain a shape to allow for activity to occur

transformation efficiency

A measure of success of the transformation experiment TE = number of transformed colonies on plate #1/amount of pDNA (in ug) on plate #1 10 uL(0.8 ug/uL) = 8.0 ug 10 uL + 250 uL + 250 uL = 510 uL 8.0 ug/510 uL = 0.016 ug/uL (0.016 ug/uL)(100 uL) 1.6 ug Transformational efficiency = 34 transformants (colonies fluoresced)/1.6 ug = 21.25 transformants/ug

dilution factor

DF = final volume/initial volume number of colonies appeared if hadn't been diluted = dilution factor x number of colonies on plate number 4

lab 6 procedure: part 2

DNA profiling: only a few cells sufficient because of amplification method. Once DNA is isolated, PCR is used to make millions of copies of small regions of the genome called short tandem repeats (STRs) STRs = short stretched of DNA that have repeated sequences, length of segment varies and difference is measurable. polymerase chain reaction (PCR) = technique that required only minuscule amounts of DNA to make billions of copies of specific targeted DNA segments. Gel electrophoresis is used to separatete the replicated STRs according to length. Shorter STRs-those with fewer number of repeats-are smaller and travel farther in the gel; longer ones do not travel as far.

Evaluate data before creating a graph

Determine which variable is independent (x) and which is dependent (y) (Independent causes a change in dependent and it isn't possible that dependent could cause a change in independent) Choose the appropriate type of graph: line, bar, pie, scatterplot

Lab 2 Procedure

I. 10, 20, 30% sucrose solutions in dialysis tubing, placed in water for 10,20,30,40,50,60 minutes, measure weight after each interval. at end measured total surface area process of osmosis using dialysis tubing as an analog of cell membrane II. NaCl (0.1,0.2,0.3,0.4,0.6,0.8 M), placed paper thin section of onion's top pigmented cell layer, left for 10 minutes, and observed under microscope of indication of plasmolysis effects of hypertonicity on red onion cells

Lab 3 Procedure

I. activity of enzyme lactase, prepared blank with lactase, Na2CO3 and ONPG. put lactase in 37˚C water baths, and added ONPG for 5,10,15,20 minutes, stopping reaction after each interval with Na2CO3. and measuring absorbance II. produced standard curve, prepared blank with 0.6 units/ml enzyme soon + Na2CO3 soon + ONPG, put 1 mL of 1,0.8,0.6,0.4,0.2,0.1 enzyme standards (units/ml) and places in 37˚C water bath for 5 minutes and then adding ONPG to start reaction. after 10 minutes, added Na2CO3 to stop reaction and measured absorbance III. effect of temp on enzyme activity, placed lactase in ice, room, 37˚C, 45˚C, 60˚C and boiling baths. after 5 minutes, added ONPG to start reaction, then after 20 minutes added Na2CO3 to stop reaction. ran blank I and recorded absorbances.

lactase catalyzes the conversion of

ONPG into galactose and ONP

substrate

ONPG, can readily bind to active site of lactase and can be broken down to produce galactose and ONP uses this because ONP is yellow, and ONPG is colorless, therefore can determine how much ONP is produced by measuring the intensity of the yellow color using spectrophotometer

lab 2 parts I & II comparison

Part one and part two both deal with the subject of osmosis. In part one, we had three different sucrose solutions (10%, 20%, 30%), and calculated the rate of osmosis. In part two, we placed a red onion's top pigmented cell layer into six solutions with various concentrations of NaCl (0.1M, 0.2M, 0.3M, 0.4M, 0.6M, 0.8M) to observe if they experienced plasmolysis. The two are parts are related in a way that as the concentration of solute increases, so does the rate of osmosis. In other words, as the sucrose became more concentrated, the rate of osmosis for the most part increased, and as NaCl concentration increased, the cell went through osmosis.

In this experiment, you dissolved .5 g agarose in 60 mL of buffer (~0.8% gel). If a group accidentally used 5 g of agarose (in 60 mL buffer), how would you expect the results to differ? Explain

The dyes would move slower, so they wouldn't travel as far

explain how plates 1, 3, and 4 serve as controls. for example what would you conclude if: a) there were colonies on all four plates? b) plate 4 was the only plate with colonies? c) the results were as expected with regard to the number of colonies but there was no fluorescence?

a) the ampicillin was defective and didn't kill any bacteria b) the transformation was unsuccessful, even plates with +pGLO didn't have colonies survive c) transformation occurred but there was a defect between the interaction of AraC and arabinose causing the GTP gene not being expressed, therefore no fluorescent

how did you determine osmolarity of red onion cells?

at concentrations 0.1 to 0.3 there was no plasmolysis meaning the solution was hypotonic to the cell and concentration 0.4 to 0.8 the solution became hypertonic to the cell causing the cell to undergo plasmolysis. so between 0.3 to 0.4 M is where osmolarity is because the cell plasmolyze within these concentrations. assuming the cell and solution reached a point of isotonic, where conc is equal inside and outside of cell

enzymes

biochemical catalysts that accelerate and steer the chemical processes in organisms are proteins whose job is to facilitate a chemical reaction, consisting of amino acids

leukemia

cancer of white blood cells cancer = cells replicate without control to replicate, cells must replicate their DNA certain proteins are needed for this process-some promote cell division, others stop it

in the electrophoresis experiment of lab 6 ______ molecules will be separated a) DNA b) none of the above c) lipid d) dye e) RFLP

d) dye

why are there differences between data sets?

differences could occur because excess water could have not been dried off completely before weight, causing masses to be different. standard error would increase because there is more time to create more errors and outlier data. standard errors show range in which data points differ from the average and can fall

standard deviation (S) and standard error (StErr)

differences in data are analyzed using these S = √(∑[(X-Xavg)^2]/n-1) StErr = s/√n ∑ = sum X = an individual value in data set Xavg = the mean of all values in the data n = number of values in the data set (sample size)

osmosis

diffusion of water across a membrane. concentration gradient for water is opposite to the gradient for solutes

osmosis refers specifically to the

diffusion of water molecules across a membrane

absorbance numbers shouldn't be above 1

dilute, run absorbance and multiply by fold

examine figure 2, what is the relationship between rate and concentration?

directly proportional, as concentration increases, rate of osmosis increase

how to calculate maximum percentage

divide the absorbance by the highest abs reading obtained and multiply by 100

increase/decrease fold

divided each drug by the control and made a bar graph

properties of enzymes result from their 3D structures

each kind of enzyme is specific for the particular reaction or class of reaction which is can catalyze because of precise configuration, which determines that only certain reacts can fit specifically into active site agents which alter the 3D config. cause a change in catalytic activity of enzyme. denaturing agents such as high temperatures or strong acids, which cause a gross untwisting and disorganization of 3D config., thus destroyed the active site and abolishing enzyme's catalytic power

given knowledge on protein structure you should be surprised by results obtained for tube in boiling water bath. explain

enzyme activity is very low, therefore it doesn't really do well in high temperatures because enzyme's active site can be altered in different temperatures so at boiling point temp. it doesn't want to bind resulting in low activity

lab 3 analysis

figure 1: line graph, as ONP production increases, time increases figure 2: line graph, "standard curve", as ONP production increases, enzyme conc increases figure 3: enzyme activity (% max absorbance) and reaction temperature, line graph, optimal temp = 45˚C

the active site of an enzyme is

fragile and dynamic, complementary in shape and chemistry, the catalytically active region of an enzyme

for which two temps was enzyme activity lowest? make sense?

freezing point (~-1.0˚C) and boiling point (95.0˚C) a high temperatures makes sense because kinetic energy increases and H-bonds could have easily been broken causing denaturation of enzyme; therefore the enzyme activity would decrease. active side can change to extreme temps allowing it to be more/less accessible

lab 6

genetic engineering: isolate DNA; long strands of DNA cut into smaller fragments with enzymes called restriction endonucleases, separated by size using a technique called agarose gel electrophoresis-allows particular piece of DNA containing a desired gene to be isolated take piece of DNA and insert into another cell DNA inserted to a plasmid (pGLO) and then transformed into a bacterial cell (E. coli); if accepts foreign DNA and beings producing protein product of gene, recipient cell engineered into a new life form

lab 7 - transformation and gene regulation

genetic transformation = change caused by genes and involves the insertion of a gene into an organism in order to change the organism's trait transforms bacteria with a gene that codes for Green fluorescent protein (GFP). following transformation. bacteria express newly acquired jellyfish gene and produce the fluorescent protein plasmid = aids with the process of moving genes from one organism to another regulation = pGLO plasmid encodes a gene for resistance to the antibiotic ampicillin the bla gene on the pGLO plasmid is always active and so cells carrying the plasmid will be resistant to ampicillin araC gene is also always active, encodes a regulatory proteins (AraC) that can bind to the promoter of genes in the bacterial chromosome encoding the enzymes necessary to digest the sugar arabinose. araC does not bind to promotor unless arabinose present in growth media arabinose controls expression of GFP gene, if arabinose is present with pGLO plasmid, AraC will lead to the synthesis of GFP and the enzymes to digest arabinose, so fluoresce

how would results differ if used 20% solution of NaCl instead of sucrose?

if use of NaCl, the mass of our tubes would increase because the water will want to flow inside at a faster rate due to it being more hypertonic that the water on the outside (molar conc NaCl = 3.45 M)

subiolsamide vs. caldribine

inhibits cell division of a number of different leukemia cell lines in a cell culture lab 1: comparing the two types of drugs to see which one was more effective

On which of the plates would you expect to find bacterial colonies made up of cells most like the original non-transformed E. coli colonies you initially observed?

plate #4 because plate wasn't transformed

lab 6 procedure: part 1

use gel electrophoresis to separate dye molecules based on charge, size and shape gel made by dissolving agarose powder in boiling buffer solution, the solution is cooled and then pouring into a gel mold, which has a template to create wells in the gel for the samples. After the gel solidifies, it is submerged in a buffer chamber that contains electrodes. Samples are prepared by mixing them with solutions containing glycerol/sucrose. This makes the sample denser than the buffer, allowing to sink and remain in wells when transferred with micropipet buffer in gel and chamber acts as an electrical circuit between the electrodes. charged molecules having a net negative charge migrate towards the positive electrode (anode) while net positively charged molecules migrate toward the negative electrode (cathode). higher the voltage, faster the sample migrates agarose is a polysaccharide derivative of agar. matrix created in the agarose gel contains microscopic pores which act as molecular sieves. sieves influence rate at which molecule migrates. smaller, compact shapes (sphere more compact than rod), greater charged molecules move through the pores more easily than larger, long, and less charged ones CHARGE, SIZE, SHAPE stain the gel, unless separated by colored dyes

Scatterplots

used to determine if two continuous variable are correlated. unlike a line graph, all data points are plotted, and a line of best fit (or tend line) is calculated and placed on the graph

in today's lab you will measure the movement of ______ across dialysis tubing

water

study III: effects on other cells (lab 1)

within the control healthy cells were dominantly present than sick and dead. While most cells were healthy for both drugs they are less than the control. But more sick cells for subiol. than clad and more dead cells from clad. than subiol. in conclusion, the control or absence of drug produces most healthy cells (BAR GRAPH)

enzymes are said to act as catalysts of reactions because they act to speed up the chemical reaction but are not used up in the reaction. does data (figure 1) support this?

yes, data supports because as time went on, even operating at 20 minutes, the amount of absorbance still increased. increase in absorbance indicates that the rate is moving faster because my slope also increased, if enzyme was used up, graph would plateau

examining figure 1, what would you expect the 30% bag to weigh when t = 2 hours? what forces eventually cause the curve to level off?

~26-27 g in 2 hours because the change in weight is about the same as time goes on. eventually, the bag would become full and the net movement of water would become equivalent causing the curve to level off