enriched material

Question 1

what is the first step to co-immunoprecipitation?

Answer 1

cell lysis

Question 1

what is co-immunoprecipitation?

Answer 1

looking for what’s attached to a protein, separation of protein, seeing if 2 proteins interact in a cell (the case we’re seeing)

Question 2

explain the procedure of co-immunoprecipiation

Answer 2

1) cell lysis
2) after cell lysis, we have A and B protein, but we also have a whole lot of other cell contents in our test tube
3) add an antibody to the mixture that will bind to protein A
4) one antibody is not strong enough, therefore we add another one that will bind to first antibody
5) we get a bead that binds to the antibodies
6) now, with our bead, we have something different to the rest of the test tube, so we can centrifuge/wash, and the bead with the attached protein and antibodies will fall to the bottom
7) boil beads in and SDS-PAGE buffer and run sample. further analyze by western blot to look for presence of protein B

Question 3

what is the goal of the co-immunoprecipitation experiment?

Answer 3

-let say we have protein A and B, and we want to see if they interact
-the goal is to fish out A and see what comes out attached (or vice-versa)

Question 4

what does the western blot experiment allow us to do?

Answer 4

visualize proteins in complex mix

Question 5

what does the SDS-PAGE experience allow us to do?

Answer 5

separate components (proteins) based on size

Question 6

what are chemolithotrophs?

Answer 6

type of bacteria that can extract energy from inorganic molecules

Question 7

what is the cable bacteria?

Answer 7

a type of chemolithotroph, that can move electrons

Question 8

how does the cable bacteria orient itself?

Answer 8

always orients itself so that one end is in an area that doesn’t have oxygen in it but has sulfides, and the other end is in an area that has oxygen

Question 9

what is the goal of site directed mutagenesis of catalytic residue? what is the procedure?

Answer 9

-figure out which amino acids are at the active site and catalyze the reaction
-we are mutating all the catalytic triad residue amino acids to alanine, to see the effect this will have on their function

Question 10

which amino acids are getting mutated during site-directed mutagenesis?

Answer 10

histidine, serine and aspartic acid

Question 11

what were the 3 amino acids mutated to during site-directed mutagenesis? why were they mutated to this amino acid?

Answer 11

allanine. they chose alanine because it was very different from the other amino acids, but as still about the same size. in fact, it is hydrophobic, therefore if the other amino acids turn into it, they will not be able to function in the active site of the enzyme anymore, and enzyme activity will decrease

Question 12

what are the properties that differ serine, histidine and aspartic acid from alanine?

Answer 12

serine: polar
histidine: charged
aspartic acid: charged

while alanine is hydrophobic

Question 13

which amino acids were determined to be part of the active site of the enzyme after their mutation to alanine? why?

Answer 13

-S80A
-H236A
-D208A

their mutation significantly affected enzyme activity

Question 14

what is km and vmax?

Answer 14

different measures of enzyme activity

Question 15

how did we determine that serine, aspartic acid and histidine were the important amino acids that we should test for, as they have a high chance of being found in the active site?

Answer 15

they determined this from previous knowledge that they had:
-chemicals that modify serine and histidine significantly affect enzyme activity
-they also know that every time a histidine and serine are involved together , aspartic acid is important for the mechanism to work

they used all this info to zero in on what’s happening in the enzyme

Question 16

what is the specific enzyme HNL enzyme that we’re testing in this experiment?

Answer 16

MeHNL

Question 17

what is a lyase?

Answer 17

a type of enzyme

Question 18

what are catalytic triad residues?

Answer 18

amino acids found in the active site of an enzyme???

Question 19

what organism is the enzyme used for the direct-site mutagenesis taken from?

Answer 19

plant

Question 20

What do the colours represent in this picture?

Answer 20

Pink: catalytic triad residue (amino acids important at active site)
Blue: substrate
Orange: other mutated amino acids that did not have an effect on enzyme activity

Question 21

what is direct site mutagenesis as a procedure?

Answer 21

a slight modification of DNA (modify one amino acid)

Question 22

what is the similarity between us an cable bacteria?

Answer 22

for the cable bacteria, oxygen is the last electron acceptor, when electrons move through it. this is the same for us with cellular respiration, as oxygen is the final electron acceptor

Question 23

what is getting reduced and oxidized with the movement of the cable bacteria?

Answer 23

-H2S getting oxidized
-02 being reduced

Question 24

what is the cable bacteria’s source of electrons? what is our source of electrons?

Answer 24

inorganic molecules. for us, its glucose

Question 25

how can cable bacteria be used to build biodegradable electronics?

Answer 25

scientists saw that the flow of electrons from one cell to another might be a source of electricity (like a battery)

Question 26

how could cable bacteria potentially be used to clean up oil spills?

Answer 26

Oil-eating microbes can clean up seafloor sediments contaminated by an oil spill. But the microbes generate toxic sulfides as waste, which hampers the microbes’ activity. Cable bacteria can help by harvesting electrons from the sulfides, converting them into sulfates, which the oil-eating microbes use as fuel. Meanwhile, the cable bacteria channel the electrons up their bodies and pass them off to oxygen in the sediment. This transport powers the cable bacteria’s metabolism in the process.

Question 27

why is there rust (orange color) at the top of the cable bacteria sample?

Answer 27

As the cable bacteria pulled electrons from sulfides, converting the noxious chemicals into less-harmful sulfates, the water within the sediment became more acidic, which dissolved some minerals containing iron. The now-mobile iron percolated upward in the sediment, until it interacted with oxygen to form rust.

Question 28

what does the top layer and the bottom layer of the cable bacteria have?

Answer 28

-top: oxygen, iron (orange colour)
-bottom: H2s, sulfate