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102 Cards in this Set

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What does protease do?
degrade protein
DNA nuclease
digests DNA eliminates transormation
recombinant DNA
inserted pieces of DNA into a vector
What is a vector?
DNA that is easy to work with and has selectable genes
5 steps to generate recombinant DNA
1. Isolate DNA - both source and vector
2. Restriction digestion - make smaller pieces of source DNA and creates a cloning site in the vector DNA
3. ligation reactions - insert vairous pieces of source DNA into the vecotr DNA
4. Transormation - uptake and epxression vector DNA into a host, typ eubacteria
5. Screen host cells fro those that have recombinant vector DNA that contains yoru gene of interests
What is a common method to isolate DNA
1. lysing cells with detergents.
2. treated with protease and RNAase
3. lysate treatd with phenol
4. dailyzed to remove low mol weight
5. ehtanol added and DNA precipiates
What is fucntion of restriction enzymes?
fucntion as immune system for prokaryotes.
what is palindromic?
each strand reads the same in 5' to 3'
What is the benefit of using restriction enzymes?
They cut at specific sites. allows scientists to manipulate and recombine DNA
What is transormation?
a way of "getting" the plasmid into the bacterium and having it expressed
Ligate
covalently link
vector DNA
typically plasmid or viral
cannot replicate itself and must find its way into a host
Advantages of vectors
list 3
1. # of base pair is few minimize damage to DNA
2. replicates independently and can be found in large #
3. contain selectable genes
What is the most widely used bacterium for vectors?
Escherichia coli
What is the most common used virus for vectors?
lambda virus
bacteriophage
virus that infects a bacterium
lytic cycle
bacterium lyse after being infected, thus releasing the viral progeny
What is the prophage stage?
when the viral DNA is incorporated into its chromosome
describe plasmids
double stranded closed circular molecules of DNA found in cytosol
What are benefits of plasmids?
anti biotic resistance
What does pBLU have a resistance for?
ampicillin
Ampicillin
antibiotic which inhibits cell wall synthesis in eubacteria.

Eubacteria containing pBLU produce protein, beta-lactamase that chemically modiefies ampicillin.
What restriction enzyme do we use in GMB I?
HinD III
What are the 3 classes of plasmid for GMB I?
1. re-ligated plasmid with no insert
2. two types of plasmids with an insert, either your gene of interest or
3. not your gene of interest
Why do we only have 2 classes in our ligation reaction?
b/c they were purified
LB
Luria Bertani Broth
contains primarily glucose as an energy and carbon source
What is the purpose of "agar" in LB?
"gels" the medium such that a solid surface is formed, and bacteeria can be immobilized onto solid medium
What are the two markers of pBLU?
ampicillin resistance and beta-galactosidase activity
What does ampicilin resistnace tell us?
differentiate btwn transformed and non-transformed
What does beta-galactosidase activity tell us?
diffrentiate between plasmid DNA with and without an insert
What are features that make pBLU an effective cloning plasmid?
1. origign of replication
2. selectable markers
(antibiotic resistance, color selection)
3. multiple cloning site
What color is the colony if the host contains ampicillin resistance?
blue
How does it turn blue?
beta-galactosidase, an enzyme conversts galactose anolog, X-gal, into blue pigment
What is the goal of the GMB lab?
To insert foreign DNA into the MCS. the insertion destroys beta-gal activity.
Common characteristics that all organisms share
list 4
1. all organsims made up of single cell or many cells.
2. cells have accurate and programmed mech for repoduction and metabolism
3. cells come from pre-existing cells on a human time scale
4. all cells are ogranized according to fund euk and prok pattern.
5 kingdoms
Monera, Protista, Animalia, Fungi, and Plantae
three domains
bacteria, archaea, and Eukarya
Archaea
membrane bound. posses nucleus which is membrane-bounded.
Waht do all organisms do similarily?
acqure energy and raw materials

transform energy and raw materials

reproduction
what two domains does prokaryotic organisms fall into?
BActeria and Archaea
autotrophic
photosynthetic
chemoautotrphic
oxidize inorganic substance for energy
mesosomes
invaginations, increase the surface area of the cell membrane
3 shapes of prokaryotes
1. cocci (spherical
2. bacilli (rod shaped)
3. spirilli (spiral shaped
gram positive prokaryotes
the cell wall retains violet color
gram-negative
red b/c crystal violet iodine is rinsed away replaced wit hsafranin red.

thin walls

tend to be more threatening
4 kingdoms that eukaryotes belong to
Plantae, Animalia, Fungi, and protists (single cell)
What is the purpose of methocel?
to slow down organisms
4 tissue types
epithelium, connective tissue, muscle tissue, and nervous tissue
Plants consist of three tissue systems
1. ground tissue system
2. vascular tissue system
3. dermal tissue system
Ground tissue consists of three cell types
1. paranchyma
2. collenchyma
3. sclerenchyma
Vascular consists of what 2 things?
xylem and phloem
heterocysts
specialized cells found in cyanobacteria responsible for fixation of Nitrogen
amylase
enzyme hydrolyzes starch
What are two functions of DNS?
1. extreme alkalinity denatures amylase
2. indirectly allows you to measure the amount of maltose
How does DNS indirectly allows to measure maltose?
DNS reacts with aldehyde groups. each maltose formed as one aldyhde group that can react.
What to add for Blank?
add amylase, then DNS, and finally starch.

DNS denature amylase b4 it can react with starch
OD units conversion
0.4 OD units = 1 micromole of maltose
affinity equation
1/Km
Km value is equal to what?
equal to the amount of substrate at whcih the enzyme is operating at 1/2 its max velocity (Vmax)
HOw do you calculate how much activity in undiluted saliva?
i dont know really
Why did we use amylase for the lab?
easy to work iwth and there are several different forms or isoszymes
Do Enzymes change delta G of reactions?
NO
What does enzyme do to the activation energy?
lowers it.
When are carbohydrates called monosaccharides? polysaccharides?
if they are made of only one simple sugar. polymers of many simple sugars
maltodextran
maltose and polymers of various sizes
phosphorylase
breaks down glycogen
beta amylases occur only in plants or animals?
plants
isozymes
have variations in both structure and catalytic characteristics
what does saliva consist of?
amylase, water, other proteins, mineral salts, and mucin
mucin
viscous glycoprotein
Why are many sugars classified as reducing sugars?
b/c they contain reactive ketone or aldehyde groups, capable of redicing certain compounds
Why is it that extremely large polysaccharide such as startch, does not act as reducing sugar?
only a few "loose ends" less ketone or aldyhde groups.
enzyme-substrate affinity
a concept which characterizes the relationsihp between an enzyme and its stubstrate
low affinity enzyme
same as low sensitivity
reaction proceeds at a respectable rate only when substrate is present in high concentration
hgih affinity enzyme
high sensitivity
enzyme reacts even if substrate concentration is low
what does low Km suggest? high Km
low Km is high affinity and high Km value is low affinity
List three major effects temperature has on enzyme catalyzed reaction.
1. mkinetic largely determines the rate for both forward and reverse rxn. temp is measure of kinetics
2. the addition of heat or removal of heat can result in one rxn rate becoming greater than the other
3. temp affect shape of proteins
is denaturation a reaction
YES!!!!!!!!!!!
T or F
the duration of exposure to high temp is not equally as significant as the actual temp of exposure.
FALSE
List two major affects that pH has on enzyme catalyzed reactions
1. may cause denaturation
2. may affect reaction rates without causing denaturation
where does carbon fixation occur?
stroma
photosystem consists of two parts
antennae and rxn center located thylakod membrane
proton gradient in cholorplast is from what to what?
high concentration (lumen) to low concentration (stroma)
NADPH and H and ATP is used to reduce CO2 to what?
CH2O by the carbon fixation reactions. Calvin cycle
How could you measure photosynthesis?
follow changes in concentration of either substrates or prodcuts. the lab you use DCPIP
Why do we use DCPIP
in vivo NADP+. it is blue, but the reduced form, DCPIPH2 is colorless.
During ETC where are the protons being pumped?
pumped from the stroma into the lumen
Where does the Calvin cycle occur?
stroma
What is the purpose of using methylamine (CH3NH2)
acts as pH buffer to minimize changes in proton concentration in the lumen
what is purpose of having sucrose?
acts as osmoticum, which prevents the chloroplasts from lysing
How do you measure the rate of photosynthesis?
measure rate of photosynthesis by measuring rate of color change of DCPIP
What are u measuring? the light or "dark" reaction?
light reaction. (PSI and PSII ETC)
List three conditions you will measure photosynthetic rates
1. darkness
2. light and DCMU
3. light
4. light and methylamine
What is DCMU?
ingredient in commerical herbicides and it blocks electron transport between PS II and PS I.
Why is methylamine used in the test for photosynthetic rates?
pH buffer minimizes changes in the proton gradient in the thylakod lumen. Without proton gradient cannot make ATP
Why doesn't thylakoid membranes rupture?
due to the presence of galactolipids
What does methylamine do to ETC?
increases it. effect is known as "uncoupling" of ATP formation form electron flow
HOw do you make absorption spectrum of purified pigments?
obtain similar band from several chromatograms and "pool" them together. add actone extract pigments. then measure aborbance every 20 nanometers.
What do acetone extracted pigments do when irradiated with UV light?
release energy as heat and fluorescence.
oxygen liberated in plant photosynthesis comes from what?
H20 dumbass!
calvin cycle forms what products?
sugar and starch