Lecture 12 Flashcards
lac operon
structural gene complex
-coregulated so all or none
what does I encode?
repressor protein that will bind to lac O and sit on the DNA preventing transcription. of the structural coding genes (YZA)
what does the inducer do?
inhibit repressor proteins from binding therefore letting ZYA transcribe to mRNA and then enzyme/ protein
-IPTG or lactose
Where does the repressor bind?
lac O
-the part next to the Z
what happens at lac P?
RNA pol will bind
what prevents RNA pol from binding to lac P?
a mutation to lac P or a repressor protein (on lac O)
mutation of lac O
-doesnt let the repressor bind therefore enzymes are constitutive
What removes the respressor?
inhibitors (IPTG or lactose)
-lets RNA pol bind
lac I-
cant make repressor therefore constitutive
-recessive to I
-trans
where does lac I come from
get it from the plasmid and genes repress lac O on plasmid and original DNA
-therefore it is TRANS
what is dom LacI^s or Lac I+
Lac I ^S
what makes lac I ^S silence the repressor/ stop enzymes
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)
PAJAMO
-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
what happens if we add inducer to PAJAMO?
we start to make enzyme again bc we are blocking the repressor protein
lac I
is an inducer regulated DNA binding protein
-forms a tetromer (4 lac I molecule complex) that binds to IPG or lactose
what happens to lac I tetromer if there is no inducer present?
it binds to lac O
Why are DNA binding proteins so fast?
it is a 1D search where they slide along the strand and stop at their target
what regulates DNA binding protein?
lactose and IPG
how can genes be regulated by glucose?
to use lactose we have to turn on genes and glucose can regulate those genes
catabolic repression
glucose stopping enzyme production
-works using adenyl cyclase and camp
what makes cyclic AMP
adenyl cyclase enzyme
-it will attach a 5 prime phosphate to a 3 prime hydroxyl making monophosphate cyclic amp
what can break down camp into amp?
phosphodiestrase
no glucose
high CAMP levels bc we are making a lot of enzymes at a steady concentration
what does glucose inhibit
adenyl cyclase
-it will lower camp because it gets destroyed by the phosphodiestrase and were not making more
CAMP binds where?
to CAP (catabolite activator protein)
-binds when active
What is CAP
A DNA binding protein
-next to RNA binding site on lac P
-will recruit RNA pol to lac P which helps start transcription
positive regulation
increase glucose, decrease CAMP, no CAP binding, no RNA pol
negative regulation
decrease glucose, increase CAMP, bind to CAP, brings RNA pol
why will the absence of lactose prevent transcription?
because there is a repressor bound to lac O that we cant remove without inducer
lactose
binds to repressor letting transcription happen bc repressor no longer blocking RNA pol
what results in very little mRNA
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
low glucose low lactose
no mRNA
shine dalgaro
where ribosome binds to mRNA
-BEFORE transcription starts
core promoter
RNA pol binding site
promoter
DNA regulator site
-includes operator
what are the repressor site and CAP site?
cis regulatory sites bc they will control the expression of the genes
recombinant DNA
DNA that is articifial/ synthetically attached
gene cloning
molecule isolation
-pure gene in a test tube
transgenic
expressing a foreign gene in a different organism
-jelly fish cell in our wormies
-rhodospin upon light exposure in our wormies
plasmid have what?
antibiotic resistant marker
-forces the cell to accept palsmid or die
-also has the replication origin
what do some plasmids have to control gene expression
control region/ promoter
typical plasmids used in the lab
F-
e.coli plasmid
Pet21
-can make transgenic bacteria
-has restriction sites
pet 21 antibiotic resistance
-ampicillan resistance
restriction enzyme
recognize and cleave DNA sequences
-leaves sticky ends for recombinant molecule
vector
plasmid
how can we make bacteria how we want it?
-clone the gene we want
-insert to plasmid
-insert to bacteria
-let bacteria replicate then isolate the gene in a test tubeh
how can we get expression vector from cloning bacteria?
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
gibson assembly
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
what makes sticky ends in gibson?
exonuclease that chews back so the PCR product can aneel to the linear vector
why is the restriction enzyme limited?
because we can only input a complementary sequence while gibson can be any sequence
what guides PCR
the primers you use
How can we cause a mutation in PCR
use a mismatch primer to make substitution
what amplifies pcr primer
end primer
calmodulin protein
binds to calcium which changes its shape allowing peptide to bind
-the protein is folded over when calcium is bound
-lets us detect calcium
calmodulin in the lab
-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
