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genetics > Lecture 12 > Flashcards

Lecture 12 Flashcards

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Biology 101 flashcards
1
Q

lac operon

A

structural gene complex
-coregulated so all or none

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2
Q

what does I encode?

A

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

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3
Q

what does the inducer do?

A

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

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4
Q

Where does the repressor bind?

A

lac O
-the part next to the Z

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5
Q

what happens at lac P?

A

RNA pol will bind

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6
Q

what prevents RNA pol from binding to lac P?

A

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

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7
Q

mutation of lac O

A

-doesnt let the repressor bind therefore enzymes are constitutive

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8
Q

What removes the respressor?

A

inhibitors (IPTG or lactose)
-lets RNA pol bind

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9
Q

lac I-

A

cant make repressor therefore constitutive
-recessive to I
-trans

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10
Q

where does lac I come from

A

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

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11
Q

what is dom LacI^s or Lac I+

A

Lac I ^S

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12
Q

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

A

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)

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13
Q

PAJAMO

A

-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

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14
Q

what happens if we add inducer to PAJAMO?

A

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

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15
Q

lac I

A

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

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16
Q

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

A

it binds to lac O

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17
Q

Why are DNA binding proteins so fast?

A

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

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18
Q

what regulates DNA binding protein?

A

lactose and IPG

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19
Q

how can genes be regulated by glucose?

A

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

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20
Q

catabolic repression

A

glucose stopping enzyme production
-works using adenyl cyclase and camp

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21
Q

what makes cyclic AMP

A

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

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22
Q

what can break down camp into amp?

A

phosphodiestrase

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23
Q

no glucose

A

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

24
Q

what does glucose inhibit

A

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

25
Q

CAMP binds where?

A

to CAP (catabolite activator protein)
-binds when active

26
Q

What is CAP

A

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

27
Q

positive regulation

A

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

28
Q

negative regulation

A

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

29
Q

why will the absence of lactose prevent transcription?

A

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

30
Q

lactose

A

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

31
Q

what results in very little mRNA

A

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

32
Q

low glucose low lactose

A

no mRNA

33
Q

shine dalgaro

A

where ribosome binds to mRNA
-BEFORE transcription starts

34
Q

core promoter

A

RNA pol binding site

35
Q

promoter

A

DNA regulator site
-includes operator

36
Q

what are the repressor site and CAP site?

A

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

37
Q

recombinant DNA

A

DNA that is articifial/ synthetically attached

38
Q

gene cloning

A

molecule isolation
-pure gene in a test tube

39
Q

transgenic

A

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

40
Q

plasmid have what?

A

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

41
Q

what do some plasmids have to control gene expression

A

control region/ promoter

42
Q

typical plasmids used in the lab

A

F-

43
Q

e.coli plasmid

A

Pet21
-can make transgenic bacteria
-has restriction sites

44
Q

pet 21 antibiotic resistance

A

-ampicillan resistance

45
Q

restriction enzyme

A

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

46
Q

vector

A

plasmid

47
Q

how can we make bacteria how we want it?

A

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

48
Q

how can we get expression vector from cloning bacteria?

A

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

49
Q

gibson assembly

A

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

50
Q

what makes sticky ends in gibson?

A

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

51
Q

why is the restriction enzyme limited?

A

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

52
Q

what guides PCR

A

the primers you use

53
Q

How can we cause a mutation in PCR

A

use a mismatch primer to make substitution

54
Q

what amplifies pcr primer

A

end primer

55
Q

calmodulin protein

A

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

56
Q

calmodulin in the lab

A

-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

57
Q
A

genetics (16 decks)

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