Bio Micro Exam 3 Slides 22-24

Front 1

What is transduction?

Back 1

This is the transfer of genetic information by a phage particle

Front 2

There are two types of transduction, general and specialized. What are the differences?

Back 2

Generalized: May involve any host gene

Specialized: occurs to genes close to the prophage

Front 3

In generalized transduction what happens (example phage p1)....this is the first step

Back 3

The phage will infect the bacterial cell.

The phage genome will then replicate to form a long concatemer of DNA, like lambda

Endonuclease will then cleave the concatemers into genome size fragments along with the host DNA.
During this time there is production of capsids and packaging enzymes.

Packaging DNA will then go into the phage particles.

The packaging of DNA by phage p1 is based on size and it selects DNA fragments of about 90kb.

Front 4

After the endonuclease has broken up the host and phage DNA, what happens in the second step after the capsid has taken in the host and phage DNA?

Back 4

There will be a second round of replication and the phage will then infect with the host and phage DNA. The host DNA will cause recombination and the phage DNA will cause phage p1 lysis.

Front 5

Describe how to apply transduction with complementing of an auxotrophic mutant

Back 5

Start with a wild-type and mutant (Phe-) strain of E. coli and some P1 phage

Infect wild type (Phe+) strain with P1

Collect lysate, filter

Some of lysate will contain genomic DNA from host strain

Use filtered lysate (phage) to infect mutant (Phe-) recipient strain

Plate infected mutant strain on minimal media (no phenylalanine available)

Cells infected with phage holding wild-type gene for the Phe- allele can repair a mutation via homologous recombination

Front 6

When using transduction with complementing of an auxotrophic mutant, what happens if a host sequence, a phe(+) sequence or a phage genome is taken up by the page?

Back 6

Phe+= phe(-) mutatn allele via homologus recombination will fix it and growth occurs.

Phage genome will give a normal infection and eventual lysis will occur

Host sequence from another part will cause no growth to occur

Front 7

What type of phage is capable of specialized transduction?

Back 7

Lambda

Front 8

Describe the process of specialized transduction by lambda

Back 8

att region of phage DNA integrates into site between galactose and biotin operons

Integrase enzyme (phage gene) mediates integration into bacterial DNA

DNA copy of virus in host genome is called a prophage

*****Also****
Induction by UV radiation leads to excision of prophage from genome and lytic cycle

Front 9

Describe the steps that are involved with excision of the prophage?
....first step (Looping – alignment of P (phage) and B (host) regions)

Back 9

Looping – alignment of P (phage) and B (host) regions

Two breaks in DNA,
P regions joined

Excised phage genome – will replicate through
bidirectional/theta then rolling circle.

Front 10

What happens when there is a double strand break in the wrong place (ds breaks occur in wrong position – one break within galactose operon, one within lambda phage genome) when excision of a prophage is happening?

Back 10

The Excised circular DNA contains part of host Galactose operon and may include structural (metabolic) genes. Also part of the prophage is left behind.

Front 11

When the improper transduction happens (ex. having part of galactose operon and some bacteria and phage genome), describe how this genetic material is packaged and transfered?

Back 11

Linearized at cos sites and packaged into phage particles

phage genome is packaged and contains portion of previous host’s GAL operon next to att site.

Part of viral genome is missing (late genes)

Lambda phage with wt galactose genes: λ GAL+

Front 12

Can an improper excision of prophage lead to transducing an auxotrophic recipient?

Back 12

Yes,
Example:
GAL- mutant can not metabolize
galactose due to mutation in
structural genes of GAL operon BUT combined with the incision of the product of the improper excision, this is possible.

Front 13

After improper excision has occured and been packed and placed into another genome. The genome will be diploid (Cell is diploid for some GAL genes heterozygous for allele which is mutated, homozygous for others). This prophage is however missing late genes and will not be able to exit lysogeny and become lytic. How is this overcomed?

Back 13

Can “rescue” by super-infection with “helper phage” (wild type lambda) containing late structural genes
This superinfection must overcome interference. Repressor is made by the GAL+ prophage, preventing expression of superinfected lambda

Front 14

What is curing?

Back 14

Loss of a plasmid is called curing. Curing can be accomplished by lack of selection or intercalating agents (Acridine orange, ethidium bromide)

Front 15

Plasmids come in the __ form of DNA.
They usually posses _____ supercoils probably due to ___ Gyrase.
Also they must have a means of ________ and they often confer some form selective advantage to host cell, but only under certain conditions.

Back 15

B
negative; DNA
Replication

Front 16

The ColE1 origin is used as the origin of replication for what?

Back 16

Commercial plasmids

Front 17

rep gene(s) coded on plasmids allow for what?

Back 17

rep gene(s) coded on plasmid allow initiation at plasmids origin of replication

Front 18

It was found that the more resistant strains of bacteria contain what?

Back 18

Restriction endonuclease

Front 19

Describe restriction endonucleases 1,2,3

Back 19

Type I – recognize specific site, but cut DNA at random(?) location far from recognition site
Type II – recognize specific site and cut within or adjacent to that site. Type II enzymes have become very widely used in molecular biology.
Type III – cleave DNA about 25bp distant from recognition site

Front 20

How do restriction endonucleases cut the phage DNA but not the host DNA?

Back 20

The endonuclease can not cleave host-methylated DNA

Front 21

Restriction enzymes will recognize and cleave a ______ DNA sequence.

The restriction enzymes will cut the _____ ______ in such that the cleavage is symmetrical. The cleavage may either leave “blunt” or “sticky” ends on the DNA.

Back 21

palindromic
phosphodiester backbones

Front 22

What is the result of ligation; creating a library?

Back 22

Take your plasmid and some foreign DNA.

Use the same restriction endonuclease on both of the DNAs

Will then mix and ligate, get two different products (mostly).

--One with self ligation or recircularization.Still only one EcoR1 site.

--One with insertion of foreign (human in this case) DNA segment
This plasmid has two EcoR1 sites with the foreign sequence in between them (an “insert”)

Front 23

Describe how you would set up the screening for a ligation done for a plasmid that might have either foreign insert or no insert

Back 23

The unique cloning site is set up so it is placed within a reporter gene (lacZ, encoding β galactosidase) such that insertion of foreign DNA will disrupt the reporter gene, rendering it non-functional.

Following ligation, the DNA will be introduced into host bacteria (a transformation reaction)

The bacteria will be plated in complex (rich) media supplemented with:

Ampicillin (or similar selectable marker, bla in this picture)

IPTG: chemical which induces transcription of the lacZ gene

X-gal commercially reagent, undergoes colorimetric change when treated with β galactosidase – changes from colorless to dark blue

Then 3 Possible outcomes for the bacterial cell will occur:
Bacteria is not transformed = No plasmid: bacteria dies from ampicillin
Bacteria harbors plasmid with no insert: grows into blue colony
Bacteria harbors plasmid with insert: grows into white colony. This colony can be selected and grown pure culture in large numbers, the insert is said to be “cloned.”

Front 24

What is insertional inactivation?

Back 24

The chance of having an insertion in the unique cloning site and maintaining an open reading frame.

Front 25

When using host cells for plasmid engineering, there are a few requirements:
No endogenous _____ ______ or ____ ______ _____.

Ability to support replication of plasmid

Susceptible to antibiotic selection

Ability to take up DNA (the plasmid) at an acceptable rate ...meaning must be ______

Must not recombine DNA insert with host genome

Back 25

restriction enzyme
β galactosidase activity
Competent

Front 26

When performing the plasmid analysis and mapping why are there three bands plasmids?

Back 26

Plasmid exists in three forms in cell, these migrate at different speeds, even though the molecular weight is the same. The three forms are:

1.Open circle or “nicked” (the slowest migrating band, but its apparent size is not easy to predict)

2. Linear (runs at 4.9kb, the size of the full length plasmid)

3.Supercoiled (runs fast, but might be slower or faster than linear)

Front 27

Describe the steps for plasmid analysis and mapping..don't describe the interpretation of this process.

Back 27

Select a colony with the foreign insert and grow under ampicillin selection

Collect bacteria and purify plasmid, eliminating protein, RNA, lipids, and genomic DNA


Digest plasmid with EcoR1 (the enzyme used to clone in this case) and other enzymes

Run products on agarose gel with undigested plasmid as control, stain with ethidium bromide and visualize DNA with UV

Front 28

Relaxed plasmids or Stringent Plasmids, which have a produce more copies or less copies?

Back 28

Stringent produce less copies

Front 29

Relaxed copies of plasmids without rop genes produce more or less copies of cells?

Back 29

More

Front 30

Relaxed copies of plasmids with rop genes produce more or less copies of cells?

Back 30

Less

Front 31

Stringent plasmids must regulate the distribution of DNA to daughter cells. This is done via:

Back 31

parS: cis acting sequence on plasmid

parB: site specific DNA binding protein encoded on plasmid (parB protein binds parS site on plasmid DNA)

parA: protein with ATPase activity, also encoded on plasmid. Binds parB:DNA complex and uses ATP to move plasmid – much of movement process not well understood

Front 32

In most cases a cell that has more than one plasmid,will have one origin of replication for the plasmid. True or false?

Back 32

False, usually has more than one.
In fact, If two different plasmids with the same origin of replication are introduced into the same cell, one will be lost.


....WHICH ONE

Front 33

Plasmids with the following origin of replications, are found in what kind of bacteria.
CoE1
RK2
RSF 1010

Back 33

CoE1-E.Coli
RK2- Gram Negative
RFS 1010- Gram Negative and Positive

Front 34

Why is a plasmid sometimes bad?

Back 34

E.Coli and Clostridium example

Front 35

How is plasmid similar to a bacteriophage?

Back 35

Both are extrachromosomal (mostly) nucleic acids, evolved to propagate through bacterial populations, using host machinery

Front 36

How can E.Coli become addicted to F plasmid?

Back 36

CcdB is a toxin. Ccdb will bind to DNA gyrase and inhibit. Well the F plasmid creates Ccda and this will inhibit Ccdb.

Front 37

What are the requirements for a vector?

Back 37

High copy number
Ease of cloning (unique restriction sites)
Minimum size
Selectable marker (e.g. beta lactamase for ampicillin resistance)