Lecture 12

Question 1

lac operon

Answer 1

structural gene complex
-coregulated so all or none

Question 2

what does I encode?

Answer 2

repressor protein that will bind to lac O and sit on the DNA preventing transcription. of the structural coding genes (YZA)

Question 3

what does the inducer do?

Answer 3

inhibit repressor proteins from binding therefore letting ZYA transcribe to mRNA and then enzyme/ protein
-IPTG or lactose

Question 4

Where does the repressor bind?

Answer 4

lac O
-the part next to the Z

Question 5

what happens at lac P?

Answer 5

RNA pol will bind

Question 6

what prevents RNA pol from binding to lac P?

Answer 6

a mutation to lac P or a repressor protein (on lac O)

Question 7

mutation of lac O

Answer 7

-doesnt let the repressor bind therefore enzymes are constitutive

Question 8

What removes the respressor?

Answer 8

inhibitors (IPTG or lactose)
-lets RNA pol bind

Question 9

lac I-

Answer 9

cant make repressor therefore constitutive
-recessive to I
-trans

Question 10

where does lac I come from

Answer 10

get it from the plasmid and genes repress lac O on plasmid and original DNA
-therefore it is TRANS

Question 11

what is dom LacI^s or Lac I+

Answer 11

Lac I ^S

Question 12

what makes lac I ^S silence the repressor/ stop enzymes

Answer 12

the repressor binds to lac O with a different shape so that the inducer can not remove the repressor by binding to it (never leaves so cant transcribe)

Question 13

PAJAMO

Answer 13

-mated I+Z+ to F-I-Z- and monitored beta gal activity
-did not introduce an inducer
-the recipient has a broken beta gal gene so even though there is no active repressor there is no enzyme made (before donation)
-after donation Z+ is not in recipient so beta gal is detected and still no repressor
-eventually I+ will make enzyme but the levels of b gal dont go down they just stay level bc production is being repressed

Question 14

what happens if we add inducer to PAJAMO?

Answer 14

we start to make enzyme again bc we are blocking the repressor protein

Question 15

lac I

Answer 15

is an inducer regulated DNA binding protein
-forms a tetromer (4 lac I molecule complex) that binds to IPG or lactose

Question 16

what happens to lac I tetromer if there is no inducer present?

Answer 16

it binds to lac O

Question 17

Why are DNA binding proteins so fast?

Answer 17

it is a 1D search where they slide along the strand and stop at their target

Question 18

what regulates DNA binding protein?

Answer 18

lactose and IPG

Question 19

how can genes be regulated by glucose?

Answer 19

to use lactose we have to turn on genes and glucose can regulate those genes

Question 20

catabolic repression

Answer 20

glucose stopping enzyme production
-works using adenyl cyclase and camp

Question 21

what makes cyclic AMP

Answer 21

adenyl cyclase enzyme
-it will attach a 5 prime phosphate to a 3 prime hydroxyl making monophosphate cyclic amp

Question 22

what can break down camp into amp?

Answer 22

phosphodiestrase

Question 23

no glucose

Answer 23

high CAMP levels bc we are making a lot of enzymes at a steady concentration

Question 24

what does glucose inhibit

Answer 24

adenyl cyclase
-it will lower camp because it gets destroyed by the phosphodiestrase and were not making more

Question 25

CAMP binds where?

Answer 25

to CAP (catabolite activator protein) -binds when active

Question 26

What is CAP

Answer 26

A DNA binding protein -next to RNA binding site on lac P -will recruit RNA pol to lac P which helps start transcription

Question 27

positive regulation

Answer 27

increase glucose, decrease CAMP, no CAP binding, no RNA pol

Question 28

negative regulation

Answer 28

decrease glucose, increase CAMP, bind to CAP, brings RNA pol

Question 29

why will the absence of lactose prevent transcription?

Answer 29

because there is a repressor bound to lac O that we cant remove without inducer

Question 30

lactose

Answer 30

binds to repressor letting transcription happen bc repressor no longer blocking RNA pol

Question 31

what results in very little mRNA

Answer 31

increase glucose, decrease camp and lactose present because RNA pol is not blocked by a repressor protein due to lactose but we cant actively call RNA pol to the site bc low CAMP

Question 32

low glucose low lactose

Answer 32

no mRNA

Question 33

shine dalgaro

Answer 33

where ribosome binds to mRNA -BEFORE transcription starts

Question 34

core promoter

Answer 34

RNA pol binding site

Question 35

promoter

Answer 35

DNA regulator site -includes operator

Question 36

what are the repressor site and CAP site?

Answer 36

cis regulatory sites bc they will control the expression of the genes

Question 37

recombinant DNA

Answer 37

DNA that is articifial/ synthetically attached

Question 38

gene cloning

Answer 38

molecule isolation -pure gene in a test tube

Question 39

transgenic

Answer 39

expressing a foreign gene in a different organism -jelly fish cell in our wormies -rhodospin upon light exposure in our wormies

Question 40

plasmid have what?

Answer 40

antibiotic resistant marker -forces the cell to accept palsmid or die -also has the replication origin

Question 41

what do some plasmids have to control gene expression

Answer 41

control region/ promoter

Question 42

typical plasmids used in the lab

Answer 42

F-

Question 43

e.coli plasmid

Answer 43

Pet21 -can make transgenic bacteria -has restriction sites

Question 44

pet 21 antibiotic resistance

Answer 44

-ampicillan resistance

Question 45

restriction enzyme

Answer 45

recognize and cleave DNA sequences -leaves sticky ends for recombinant molecule

Question 46

vector

Answer 46

plasmid

Question 47

how can we make bacteria how we want it?

Answer 47

-clone the gene we want -insert to plasmid -insert to bacteria -let bacteria replicate then isolate the gene in a test tubeh

Question 48

how can we get expression vector from cloning bacteria?

Answer 48

have bacteria make protein and harvest it -increase protein production by adding IPG3 to cells so 50% of the proteins can be the ones u want

Question 49

gibson assembly

Answer 49

assemble PCR product into plasmid wihout restriction enzymes -use the pcr to make a linear vector and the destination vector will be identical to the PCR product -amplify DNA -put pcr product into the linear destination vector -making a plasmid -assembling DNA fragment

Question 50

what makes sticky ends in gibson?

Answer 50

exonuclease that chews back so the PCR product can aneel to the linear vector

Question 51

why is the restriction enzyme limited?

Answer 51

because we can only input a complementary sequence while gibson can be any sequence

Question 52

what guides PCR

Answer 52

the primers you use

Question 53

How can we cause a mutation in PCR

Answer 53

use a mismatch primer to make substitution

Question 54

what amplifies pcr primer

Answer 54

end primer

Question 55

calmodulin protein

Answer 55

binds to calcium which changes its shape allowing peptide to bind -the protein is folded over when calcium is bound -lets us detect calcium

Question 56

calmodulin in the lab

Answer 56

-fuse it to GFP and attach to peptide that calmodulin normally binds to -results in a protein that becomes fluorescent when calcium binds to it -calcium sensor -indicate if a neuron is active