fuck my fucking chungus life

Bacteria are ideal candidates for genetic research because???

of their relative genetic simplicity

Since bacterial cells have one copy of each gene, any change in that gene may produce???

a change to the observable phenotype

Lederberg experiments:

single, double, and triple mutant methionine and proline

auxotrophs were cultivated together.

In the Lederberg experiments, Progeny could survive in medium containing neither

methionine nor proline,

This meant __________

that either the exchange of genetic material between the

parental strains or reversion had occurred

Reversion

a genetic alteration that reverses the effect of a mutation;

rate of reversion

10^6 to 10^7 for a plate containing 10^8 cells

rate of reversion is minimized though the use of?

double and triple mutants

How many chromosomes do most bacteria have?

one circular

Which bacteria contain linear chromosomes?

Streptomyces, Borelia

Some bacteria contain ________or ______

chromosome or plasmids.

Unlike most Eukaryotic DNA, most bacteria is??

transcribed

What plays signficant roles in the evolution of bacterial chromosomes?

Bacteriophages and plasmid DNA

How does bacteriophages and plasmid DNA play significant roles in the evolution of bacterial chromosomes?

they provide mechanisms for the transfer of genetic material between bacterial cells

What does a mutant refer to?

a cell or a strain possessing a change in its DNA sequence (compared to its wildtype)

How can mutations be detected through?

visible changes in phenotype or growth patterns

allele

one of two or more alternative forms of a gene that arise by mutation

One method to detect mutants includes???

the use of a growth medium that does not support the growth of microbes lacking the desired gene

(antibiotic selection, duplicate plates)

What is the set up for duplicate plates?

– The first is under full

nutritional support.

– The second lacks a

particular nutrient.

Duplicate plate

Where a colony grows on the fully supported plate, but

doesn’t grown on the partial support plate, a mutation

has occurred.

What is the set up antibiotic selection?

An agent (antibiotic) is added to the medium to kill non-resistant cells;

Only resistant strains survive; non-resistant strains fail to grow

How are duplicate plates created???

replica plating

What did Esther Lederberg use in his experiments?

replica plating

Why did Esther Lederberg use replica plating?

to illustrate spontaneous mutation without

selective pressure

What did Richard Lenski do?

he analyzed the evolution of E.

coli in a culture over a period of 75 days;

What did Richard Lenski’s results from his e.coli experiment discover?

an enhanced fitness of the microbes (e.coli)

genome

all the genetic material present in a cell (including in plasmids)

replicon

is a region or unit of DNA that replicates from a single origin of replication

Can incompatible plasmids stably exist within a cell population?

no; incompatible plasmids CANNOT stably exist within a cell population

What is an example of incompatible plasmids?

if they have similar origins of

replication)

How do you write a gene?

lacY

How do you write a protein?

LacY

What do restriction enzymes act as?

molecular scissors

What do restriction enzymes (molecular scissors) do?

They recognize a specific DNA sequence and make double-stranded cuts to generate sticky ends

How long is a restriction site?

usually six to eight base pairs long

Restriction site sequences are usually _________

palindromic

What can similar ends of cut DNA do??

pair together

What can paired ends of cut DNA do?>

be tied, or ligated, by DNA ligase.

Paired or blunt pieces of DNA generated by restriction enzymes can be ligated by ???

DNA ligase,

What does DNA ligase do?

reforms phosphodiester bonds between adjacent 5’

phosphate and 3’ hydroxyl groups

When can Recombination DNA molecules be generated?

when two DNA fragments containing identical restriction sites are digested by a restriction enzyme and the resulting DNA pieces with single-stranded ends are complementarily paired;

The molecules that result from when two DNA fragments containing identical restriction sites are digested by a restriction enzyme can be used to ???

clone a bacterial gene of interest

What are vectors used for?

to insert a recombinant DNA molecule into a recipient host bacterial cell

What are examples of vectors?

plasmids, phages, cosmids),

Plasmid cloning vectors were first used by Cohen at Stanford. What were the steps?

Cut fragments from two

plasmids carrying

antibiotic resistance

genes with the same RE,

followed by ligation with

DNA ligase

– After inserting the

recombinant plasmid

into bacteria, the strain

exhibited traits from

both plasmids.

(basically, fragements from two plasmids were taken, tied together, and then inserted into a bacteria; the bacteria had traits from both plasmids)

What does the blue-white screening method do for plasmids?

It allows for visual differentiation of colonies

that carry inserts from colonies that do not

Where does the amino terminal fragment come from?

the plasmid)

Where does the carboxyl terminal fragment come from?

the E. coli)

What does the combination of the amino-terminal fragment (from the plasmid) and the carboxyl-terminal fragment (from the E. coli) of the enzyme beta-galactosidase produce?

a functional enzyme that cleaves the substrate X-gal, causing the formation of blue colonies

What does the enzyme resulting from the combination of the amino-terminal fragment (from the plasmid) and the carboxyl-terminal fragment (from the E. coli) of the enzyme beta-galactosidase cleave?

substrate X-gal

What does the enzyme resulting from the combination of the amino-terminal fragment (from the plasmid) and the carboxyl-terminal fragment (from the E. coli) of the enzyme beta-galactosidase cause?

blue colonies

What does the introduction of foreign DNA into one of the restriction sites on the plasmid do?

It disrupts the alpha fragment-encoding sequence, thereby abolishing beta-galactosidase activity and causing the

formation of a white colony

The introduction of foreign DNA into a restriction site on a plasmid disrupts alpha fragment encoding sequences.

Thiis causes the formation of what?

white colonies

What are examples of desirable plasmid cloining vectors?

origin of replication,

a selectable marker gene

multiple cloning sites,

small size,

high copy number;

What capacity is a desirable trait in a plasmid cloning vector?

15kb

What can also be used as a cloning vector?

Phage lambda

What does the phage lambda do?

takes advantage of the natural ability of viruses to infect and efficiently deliver their genomes into cells;

What is the capacity of phage lambda?

24kb

What do cosmid vectors do?

they are are phage genomes that omit nearly all of the phage DNA

Cosmid vectors leave more room for what?

the DNA fragment of interest;

What is the capacity of cosmid vectors?

35-45 kb

What does horizontal gene transfer involve?

the movement of DNA between microbes

What does recombination involve?

the incorporation of foreign DNA into the host cell’s

chromosome

What happens in homologous recombination?

two (nearly) identical fragments of DNA align and

exchange pieces;

What are the Holliday junction formation and resolution mediated by?

RecBCD and RecA in E. coli.

In homologous recombination, what does the type of molecule determine?

it determines the outcome (DNA swap

versus integration)

If there is a single crossover of two circular moledules, what does that result in?

Integration

If there is a double crossover of two circular molecules, what does that result in?

DNA swap

If there is a double crossover of circular and linear molecule, what does that result in?

Integration

What happens in non-homologous recombination?

recombination of DNA pieces with little to no

sequence similarity.

What is non-homologous recombination prevalent in?

viruses

What do temperate bacteriophages do in non-homologous recombination?

insert their

genome at particular points in the host cell

DNA sequence

In non homologous recombination, temperate bacteriophages insert their genome at particular points in the host cell

DNA sequence.

This process is facilitated by what enzyme?

integrase

What is transformation?

the introduction of extracellular DNA directly into an

organism;

Does transformation require cell-to-cell

contact?

No; transformation DOES NOT require cell-to-cell

contact?

Which bacteria is naturally competent for transformation?

Streptococcus

Which bacteria is NOT naturally competent for transformation?

E. coli

How do bacteria that are NOT naturally competent for transformation achieve artificially induced competence?

Treatment with ca 2+ (electroporation)

What do naturally competent bacteria

for transformation have for the uptake of DNA?

specialized receptors (DNA-binding receptors) and transport systems (channel-forming specialized pore proteins in the plasma membrane)

What specialized receptors do bacteria that are naturally competent for transformation have?

uptake of DNA

What is conjugation?

the transfer of DNA from cell to cell via direct contact or sex pilus formation

What is the fertility factor F?

is a circular dsDNA plasmid.

What do F + strains do?

F+ strains can donate DNA to F strains and convert these cells to F+ cells

What are F plasmid genes required for?

the production of conjugational structures that link two cells

together and facilitate DNA transfer

What do conjugational structures do?

link two cells together and facilitate DNA transfer

Can the transfer of an F plasmid integrated into the host chromosome take place?

Yes; the transfer of an F plasmid integrated into the host chromosome take place

What are the instances of the F plasmid integrating into the host chromosome?

1) Hfr (high

frequency of recombination) strain DNA

transfer.

2) generation of F'

(F prime) plasmids.

The HFR can result in the transfer of attached chromosomal genes, but what does it NOT result in?

conversion of the recipient cell to F+

Why does Hfr not result in the conversion of the recipient cell to F+?

mating pairs often separate before the transfer of the

complete donor chromosome can take place

What happens in the generation of F’ prime plasmids?

F’: the incorporated F plasmid excises itself along with some host cell DNA and, during future conjugation events, transfers host cell DNA to the recipient

When does F prime transfer host cell DNA to the recipient?

during

future conjugation events,

When does a mating bridge form?

Once contact between a donor and recipient cell has occurred,

What does a endonuclease associated with the mating bridge make?

a single-stranded nick at the oriT of the F

plasmid,

The single-stranded nick of OriT of the F plasmid made by the endonuclease allows what?

donation of a single strand of the plasmid to the recipient cell

What is Transposition?

the movement of DNA via

mobile genetic elements

Where are transposable elements move, located and occur?

genomes

What do Simple transposable elements do?

only encode

proteins for transposition