BG 3 Lytic Bacteriophage

binding of the phage to a specific receptor on cell surface

adsorption

early genes transcribed after injection of dna are

encodes mostly enzymes for dna synthesis

mrna is trancribed from ____ genes- encode proteins involved in ____

late, assembly of heads and tails (structure proteins)

how are phage genes regulated

transcription and post transcriptional regulation

regulatory cascades can easily identify a mutation how

able to see how one gene effects another. if something is mutated, you can see what’s being regulated more/less

in T7, RNA polymerase is a ________ gene. it recognized _______ promoters

late

what is the purpose of the pet system

to use T7, a pET vector, and IPTG to make large amounts of the target gene in the vector to produce lots of protein

which promoter is utilized in the pet system to make the pet vector

head protein 10

what transcribes the pet vector

t7 rna polymerase

what is needed to make the t7 rna polymerase to make more protein

a lac promoter, IPTG

how does iptg induction work

it mimics lactose and turns the system on, messing up repressor

where is your gene of interest inserted in the pet system

downstream of t7 promoter

if there was no lac operon in the pet system,

the lac operon keeps things off and controls production of t7, so if it wasn’t there, t7 would be made constantly

how would u use the pet system to ensure you get a lot of protein

let cells grow for a while, then induce with iptg so that all of those masses of cells will grow more

stop mutations can be used to identify gene products that ________ late genes

turn on

the promoter of head protein 10 in e.coli encodes what

a capsid, that makes it strong and protects it. helps it make thousands of genomes

proteins are separated by ______ in seeing infection times

gel electrophoresis

the phage polypeptide product runs towards its _______ on electrophoresis

molecular weight

early genes will be expressed on the ______ band with ______. and late genes willbe expressed on the ____ band with _____

first, late disintegration. last band buildup

why would two bands on GE be missing if there’s a stop mutation in a gene

one could possible regulate the other

what is the difference between a protein and the same protein with an asterick

they both came from one large protein, and it was eventually cleaved

in T4 genes, mutations in ___ and ))))) prevent middle genes

motA and asiA

mutations in genes ___ and ____ in T4 dna rep prevent late genes

33, 55

in t4, delayed early genes are transribed from

normal sigma 70 promoters

rna polymerase will stop before it transcribed delayed early genes unless

t4 produced antitermination factors, whcih are products of immediate early genes

transcripiton of promoters for middle genes are encoded by

motA and asiA

products of late genes

head, tail, enzymes to lyse the cell

how is the late gene promoter different than a usual promoter

it has a tataaata sequence and they lack a -35 sequence

what does gene 55 do in T4

alternative sigma factor taht binds to rna polymerase and causes it to recognize the true late promoter

the phage has its own proteins to make ______ translate it. you need ___ ____ and ____ to get late genes

rna polymerase. gene 55, gene 33, gp45

mota and asia on the sigma 70 MIDDLE GENE PROMOTERS give. _____ -35 sequence and a ______ -10 sequence

longer, normal

the late promoter has ____ -35 sequence and a ____ -10

no, longer

making things in lytic bacteria is based on

promoter recognition

phage replication is not correlated with ______

cell division

how do phages stop the primer problem from the lagging strands

telomeres, primers, terminal redundancies, and hairpin ends (concatamers)

what is the purpose of concatamers

make more dna, don’t lose genetic info, ensures full genomes are packed

in t7 concatamer formation, replication begins at the ori and replicates toward both ends while leaving _______

3’ ends because you can’t prime them

t7 has ____ at the ends of the dna so that they can ______ and form the concatamer

terminal redundancies, complement

in t7 concatamers, genomes are cut out at ______ sites and packed into ____

pac, phage heads

t4 encodes all of its ______ proteins, and contamers form in ____ stages

replication, two

in stage 1 t4 concat, it leaves _____ ends. in stage 2, it forms _____ to prime replication to make large branches. this is called

3’, loops. recombination-dependent replication

t4 is cut out of concatamers in _____. terminal redundancies are created since each t4 phage holds ____ more than the length of the genome

headfuls. 3%

in t4, each molecule cut off contains sequences of

the next genome

cyclic permutations

shifts all elements of a set by a fixed offset, so you’re getting dna from the next genome too. this is why the genetic map is shown as circular for t4. there’s no SPECIFIC start and stop

high moi means

more phage

to do phage crosses, you have to first

isolate mutant phage with desired mutations. mutated dna of the same species can be put together in the same cell for crossing

for phage crosses, the two dnas _______, after you infect the saem cell with different strains of the virus at the same time

recombine

how can we ensure that most of the cells get both viruses

there needs to be a high moi

for phage crosses, you’ll end up with ______ of one mutation, _______ of the other, _____ in two heads

parental, parental, recombinants x 2

why does complementation matter

sees if two muations can complement under conditions nonpermissive for one alone

in complementation tests, if both mutations are in the same gene, you’ll get _______. it’s good if the mutations are in _____ genes so that one can be ____

no phage. different, expressed

recombination tests are done under _____ conditions. combination tests are done in ______ conditions

permissive, nonpermissive

rII genes are

rapid lysis mutatnts type 2

r2 genes make a cell ____ quicker. they cannot multiply in strains of _____ taht are lysogenic for lambda prophage

lyse. e. coli

rII cannot multiply on

K lambda phage

recombination of rII was shown through

injecting e. coli with two different rII mutatns and letting htem multiply then plating them to see frequency of recombinatin

if recombination occurs with the two rII mutants what will you get

double mutants with both or wild type

total number of recombinant types is _____ x 2

wild type r because it grew on k lambda

how do you ensure e. coli that’s being plated on doesn’t die

need to do serial dilutions

why do you dilute total progeny more than when diluting r+ recombinants

you’ll account for recombinants and wild type

if you have 108 plaques on e. coli k lambda plates, what do you do to find total recombinants

108 × 2 = 216 × 10^5 (dilution)

if you have 144 on e. coli, how do you get total progeny

144 × 10^7

recombination frequency equation

recombinant progeny/total

multiply RF by ___ for map units to see how close together mutants are

100

to calculate moi, divide

phage added/bacteria present

high moi means

phage outnumber cells

how can poisson distribution be used for moi and what does that tell us

use e^-moi then multiply times 100 for percentage UNINFECTED

what if one parent strain had both mutations and the other had neither?

none, both, right, left

how do terminal redundancies help the primer problem

it acts as a buffer so that even if a few nucleotides are lost in replication, it’s nothing important.

t7 linear dna doens’t _____, just forms concatamers through

cyclize, joining end to end

t7’s terminal redundancies _____ and form the concatemer

pair

how do the phage heads get packed dna in t7 concatamers

cut at pac sites at the ends of dna and put in