Bio Lab 1 Final

Front 1

· Whatwas the purpose of doing the cyanobacterial project?

Back 1

Thereare 200 isolates that have not been identified. They cannot be identified untilthere is phylogenetic evidence that they are distinct. We are seeing if any ofthem are new to science.

Front 2

· Whyare cyanobacteria important?

Back 2

- oxygenate the atmosphere. In most environmentsthey fix nitrogen into ammonia. Very important for ecosystems. Source of pharmaceuticalsand biofuels. Known toxin. Stabilize soils against erosion.

Front 3

· Whyis the 16-23S r RNA region a good one to use for phylogenetic studies?

Back 3

becauseall cellular organisms have this gene, 2. it is a housekeeping gene- maintainscellular metabolism. This sequence can be compared between 2 organisms. Thiswill allow us to compare taxa. All prokaryotes have it and it is highlyconserved. Eukaryotes keep it in the mitochondria, plants have it inchloroplasts.3. It has hypervariableregions (regions where it is not as tightly conserved), This allows us tocompare closely related taxa, such as genera in a single family or species witha genius. 4. Most extensively researched region in the NCBI which allowscomparison of new taxa with a large number or organisms

Front 4

· Whatis phylogeny? Could you recognize parts of a cladogram?

Back 4

- the evolutionary history of a species or a groupof species. The more DNA in common the more closely it is related. Phylogenetictree. Node- common ancestor. Branches are what stems off of them, Membersbelonging to the same ancestor are a clade.

Front 5

· Whatare the two nucleic acids?

Back 5

Deoxyribonucleicand Ribonucleic Acid

Front 6

· Whatis the function of DNA?

Back 6

- Nucleic Acids. Function- to store and transmithereditary info. Double stranded polymer (like proteins and polysaccharides).Has information for its own replication. All living things have DNA their orderand number of nucleotides differ.

Front 7

· Whatare the building blocks of DNA called?

Back 7

-nucleotides

Front 8

· Whatthree molecules comprise these building blocks?

Back 8

-monomers?Sugar-deoxyribose. Nitrogenous base(ATGC). Phosphate group

Front 9

Whattype of bonds holds the nitrogenous bases together as the “rungs” of the helix?

Back 9

- hydrogen bonds

Front 10

· Thesugar phosphate backbone is held together by what type of covalent bonds?

Back 10

-phosphodiester bond

Front 11

· IfDNA is chemically the same in all organisms, what makes individuals different?

Back 11

- order and number of nucelotides differ

Front 12

· Whatis PCR?

Back 12

-polymerase chain reaction. Amplify (copy) DNAoutside of a cell.

Front 13

· What4 components need to be in a PCR reaction for it to proceed and what is therole of each?ron:~

Back 13

1. Buffer solution- gives thecorrect environment for DNA amplification (gives it a right/normal pH, adds Mg,2. Nucleotides- buildingblocks of DNA3. DNA Polymerase (TaqPolymerase)- heat stable, adds nucleotides, template to be copied4. DNA primers- short-singlestranded pieces of DNA that will define the region to be copied. Anneal to the3’ end

Front 14

· Whatis special about the enzyme used in the PCR reaction? Why do we need it to beheat stable?ns:~

Back 14

Heatstable and is not destroyed at high temperatures. So it will not denaturebecause increase in temperature could cause it to denature2b

Front 15

· Whatdoes a thermal cycler do?edia2~

Back 15

-rapidlycycle temperatures used to incubate the PCR reactionure c+}

Front 16

· Howdoes PCR work? What happens in thedenaturation, annealing and extension (DNA synthesis) stages?dDest$~

Back 16

Firstit is denatured (heat) then anneal (cool) and extend (warm 75 degree)

Front 17

How is a gel made? From what material?"

Back 17

- 1% agarose. Pour agarose into a buffer likejello and water. The agarose is then poured into a try and a comb is pulled outto form the wells. The gel is placed into an electrophoresis tank and iscovered with a buffer solution that will carry a currentCovered with a buffer so an electrical current canbe carried through the tank.

Front 18

Where is the DNA sample initially placed in the gel when you load it (think about where the electrodes are)? Could you set-up the electrophoresis apparatus correctly?LOR",`U~

Back 18

- On the negative well because is have a negativeelectrode (cathode) and travels to the positive electrode (anaode)e$p

Front 19

Why is the loading dye added to a DNA sample before it is put on the gel? yes'>Sf~

Back 19

1. Contains a heavy substance that when added tothe DNA substance it will become dense (glycerol)- 2. Contains a tracking dye that will travelapproximately the same rate as the DNA moving through the gel DT]

Front 20

How is the gel stained? What are the two differentstains used? What are pros and cons ofusing each? Which one did we use in lab?ause i

Back 20

- With ethidium bromide + can detect small amountsof DNA band, - known carcinogen. Used this-Methylene blue + safe to handle – need more DNAin a band to detect it. Not sensitive to DNAUG]

Front 21

Howis the distance migrated of the DNA bands determined? (|

Back 21

- by the number of base pairs in a sample. Measurefrom the marker/ standard on the side. Larger DNA move smaller amount

Front 22

What force moves the DNA through the gel?

Back 22

-electricity

Front 23

How is it that a mixture of DNA fragments of different sizes be separated on a gel?html\g~

Back 23

-Smaller pieces of DNA move father because theycan fit through the pores better than the big pieces. Higher % of agarose the smallerpore. More agarose the less space there is. Bigger pieces have bigger basepairsh

Front 24

What are DNA size standards and why do we need them on the gel? What other names do size standards go by?lected`c~

Back 24

- fragments of known DNA and sizes. Need them tomeasure the distance migrated. Standard/marker/ladder%s

Front 25

How is a standard curve made? If given a standard curve could you determine the size of a DNA fragment if given the distance migrated?sizɨhk~

Back 25

- need to know 1/ size of DNA band in marker and2. Distance each standard band has migrated from the origin or well- use equation of a standard curve. Distancemigrated is x, MW or length is y….forms a C shape GU]

Front 26

How did you know if your PCR reaction was successful? How did the results of gel electrophoresis tell you?˨jm~

Back 26

- Bubbles at the end of the comb/ wells. It wassuccessful if you see base pairs on gel. If the dye stained it and you seesomething then it didn’t work

Front 27

Whatis molecular cloning?$p

Back 27

- Using bacterial cells (E.coli) to make copies of DNA fragment of interest using a vector and recombinant plasmid. -DNA of intrest is inserted into the cloningvector (plasmid)- Bacterial cells (e.coli) take up the recombinantplasmid-Plasmid containing DNA of interest is copied bybacterial cellsNA)~

Front 28

What specific bacteria did we use to do the cloning?>

Back 28

E.Coli

Front 29

What are plasmids? How can they be used for cloning? What are some features of the plasmid we used?

Back 29

- small circular extrachromosomal DNA found inbacterial cells- DNA exists outside of chromosomes. Contain non-essentialgenes. Have antibiotic resistance genes. Bacteria can have multiple copies ofplasmid per cell. ADD MORE. HOW CAN THEY BE USED FOR CLONING—insert DNA into aplasmid. Plasmid is a vector- vector is a molecule of DNA that canshuttle/carry DNA sequences into the bacterial cells Bacteria we are using is treated with CaCl becauseit is positiveily charged calcium ions will allow negatively charged DNA topass into bacterial cell with a quick heat shock. ]

Front 30

What is bacterial transformation?:p><H}~

Back 30

- Transformation occurs when bacterial cells takeup DNA from the outside. Bacteria we are using are treated with CaCL SLIDES'q

Front 31

If bacterial transformation is an inefficient process, how do we find the relatively few bacterial cells that take up a plasmid?

Back 31

- Miniprep. Choose ampicillin resistant colony.Bacteria multiply overnight. Use a set of techniques to break open bacteria andisolate plasmid DNA(|

Front 32

What is the purpose of a miniprep? What are the roles of lysis buffer, neutralization buffer and a spin column?o

Back 32

- purpose is to make copies of the DNA if interestfor sequencing - use a set of techniques to break open bacteriaand isolates plasmid DNA - lysis buffer- breaks open bacterial cells- neutralization buffer- allows the lyse to breakDNA open until a necessary pointSpin column- DNA attaches to the spin column sothe DNA can be extracted. WHY Silica will bind to the nucleic acids undercertain conditions6'

Front 33

After isolation of the plasmid DNA, how did you confirm that the plasmid was isolated and it contained an insert of cyanobacterial DNA?

Back 33

- add EcoR1. Cut plasmid with restriction enzymethat can “cut out” the inserted DNA- restriction enzymes cut double-strandedDNA by recognizing a specific sequence and only making a cut at that sequence.2 bands on gel- 1 for the plasmid (4300 base pairs long) and one with the 1600base pair PCR product. PLASMID DNA WILL BIND TO SILICA (GLASS) MATRIX OF THECOLUMN. indeɭmQ'

Front 34

What is activation energy? How can an enzyme help lower it (how do enzymes work)?~Xm~'

Back 34

- enzymes are protein molecules that catalyzebiochemical reactions in all living organisms. Activation energy is the minimumenergy that is required to start a chemical reaction. It speeds up the rate ofchemical reactions by lowering the activation energy needed for the reaction toproceed. It can only increase the rate of reaction that would otherwisespontaneously occur. LOOK AT GRAPH AND NOTES̭h\'

Front 35

Enzymes are what type of biomolecule? enzymesSf~'

Back 35

- proteins entֻ'

Front 36

What are some properties of enzymes?Ty~'

Back 36

- They are highly specific, they are not changedby chemical reactions which means that little enzyme is needed They increasethe rate of reaction which causes the reactants and subtrates to bind faster toeachother. Has a specific active site. evel4]a'

Front 37

Why are enzymes said to have specificity?y~'

Back 37

- they only have 1 or a group of relatedreactions. Acid phosphatase from wheat germ extractmAu'

Front 38

Why is the three-dimensional shape of an enzyme important?

Back 38

- There is a recognition between the enzyme andthe substrate based on their 3D shape. An E-S complex forms. This configurationmakes el-tabK'

Front 39

What is an active site?’t;fUx~'

Back 39

- the place where the substrate and enzyme cometogether to form products. - factors that affect enzyme activity is temperature,pH, concentration of enzyme, concentration of substrate, concentration ofproducts, presence of inhibitors or activatorsent:-.2έj^'

Front 40

How can the rate of an enzymatic reaction be measured?"'>Why Qd~'

Back 40

- You can measure the rate of reaction by applyingthe right substrate, enzyme and favorable incubation conditions. Set up areaction tube (in vitro) that has the amount of substrate and enzyme desired.Measure1. the amount of substrate that diminishes overtime2. the increasing amount of product formed overtime. Youcan compare this to time0:-'

Front 41

In general, what happens to the rate of an enzymatic reaction over time?_b~'

Back 41

Asthe time the enzyme and substrate reaction increases, the the amount of productincreases. The amount of product formed is also dependent on enzyme concentration.As time increases, products increase. Low temperature slows, higher temperaturespeeds it upmber-ʭnR'

Front 42

Whatenzymatic reaction does acid phosphatase catalyze? Reactants? Products?incAu'

Back 42

- acid phosphatase removes a phosphate group underacidic conditions. Turns yellow when product was formed. Converts nitrohenylphosphate (substrate) to phosphate and nitrophenol (products). Substratenitrophenyl phosphate is colorless, when one of the products (nitrophenol) is ayellow colored compound under acidic conditions. Relationship of absorbance andconcentration-inɭmQ'

Front 43

What is a reaction tube?/ul>Ux~'

Back 43

usea spectrophotometer to measure the intensity of the yellow color. Compare to astandard ADD MORE. Is this how you perform an enzyme assay?}_c'

Front 44

What was the purpose of KOH in the enzyme assay?

Front 45

How are absorbance and concentration of product related? Could you use a standard curve to convert absorbance to concentration?Xm~'

Back 45

Absorbanceand concentration are measure together. As concentration increases, absorbance increasestext-inRf'

Front 46

In general, what does changing the temperature do to the rate of an enzymatic reaction? >]`~'

Back 46

- it can cause it to unravel, denature, slow itdown, stop forming products, bonds are broken which can cause the active siteto change. If the active change is too big then the substrate cannot bind tothe enzymeexֺ'

Front 47

What is enzyme denaturation? How can it happen?

Back 47

-Denaturation- a structural change in a protein that results in the loss of it’sbiological features. Ways to be denatured: changing of pH, extreme temperature,enzyme/substrate concentration mso­fj'

Front 48

What was the purpose of having the yeast in the respirometer?oQd~'

Back 48

The yeast converts small amounts of sugars inthe dough to carbon dioxide and alcohol. Carbon dioxide trapped in the doughmakes it rise. The alcohol evaporates in the oven. Fermentation is the name of the process that the yeast uses to make carbon dioxide.. Measurethe amount of ethanol formed and the amount of C02 given offProduct offermentation that displaces the yeast-sugar solution can be measuredt-id~'

Front 49

Why was the sugar needed in the respirometer?dSf~'

Back 49

Allows the displacement tobe measured and is needed to take place without oxygenl-Vz'

Front 50

How was the fermentation rate measured? Specifically what product were you measuring? e

Back 50

-Measure C02. Compare concentration before and after. Subtract before-aftermber-pW{'

Front 51

Besides a small amount of ATP, what are the products of glucose alcoholic fermentation?n:yes'>]`~'

Back 51

ethanoland carbon dioxideLp'

Front 52

What are the three major parts of aerobic cellular respiration? What does each yield and where do they occur?

Back 52

glycolysis-yields 2 ATP and 2 NAD, in cytosol/cytoplasm

Kreb’s cycle-yields 2 ATP, 6 NADH, and 2 FADH in mitochondrialmatrix

electron transport chain-yields 26-28 ATP in inner mitochondrialmatrix. No ATP, 10 NADH,2 FADH-

Front 53

What are the electron-carrying molecules that transport electrons to the ETC?m_b~'

Back 53

6NADH and 2 FADH per glucose molecule

Front 54

What is the difference between substrate-level and oxidative phosphorylation?m_b~'

Back 54

- Oxidative phosphorylation-involves the electron transport chainand chemiosmosis accounting for most of the ATP synthesisSubstrate level phosphorylation-directtransfer of a phosphate group to ADP from a substrate in glycolysis or citricacid cycle-id~'

Front 55

What is the ultimate electron acceptor in the ETC in aerobic cellular respiration?

Back 55

oxygen

Front 56

What are some ways fermentation differs from aerobic cellular respiration?-lef_b~'

Back 56

Fermentation does not need oxygen, and breaksdown glucose to lactic acid or ethanol and CO2. Aerobic respiration breaks downglucose to CO2 with release of 36 ATP. Fermentation releases only 2 ATP permolecule of glucose..5in;ޭzN'

Front 57

chemiosmosis

Back 57

production of atp using the energy of the proton gradients across the membrane to phosphorylate ADP to ATP

Front 58

Oxidative Phosphorylation

Back 58

the metabolic pathway in which the mitochondria in cells use thier structure, enzymes and energy released by the oxidation of nutriuents to reform ATP

Front 59

wave lengths important for photosynthesis

Back 59

chlorophylls absorb red and blue

reflect green and yellow

carotenoids absorb blue

reflect yellow and red

Front 60

what is PAR

Back 60

the amount of light available for photosynthesis, which is light in the 400 to 700 nanometer wavelength range. PAR changes seasonally and varies depending on the latitude and time of day