Exam 2- Microbial Genetics

Phage display: (a) what can it be used for? (b) why is it a powerful technology? (Practice Exam 2)

(a) To identify short protein sequences that bind to targets of interest, such as proteins (b) Because large number of clones can be easily screened for high affinity binders

What cellular/environmental factors might influence a phage's decision to lysogenize or not? Explain (Problem Set 7)

1. The number of phage in the environment If many phage are already present why make more phage? 2. The health of the cell. Healthy cells (fast growth) suggests the cell can support making a lot phage. A sick cell cannot make a lot of phage, so lysogenize and wait for another day. 3. Others?

What specific cis element does Cro have the highest affinity for? (Problem Set 7)

OR3 in the C1 promoter

What advantage(s) do lysogenetic phage have over lytic phage? (Problem Set 7)

They do not have to kill the host. Thus they can lie dormant and propagate themselves with the host. In addition and importantly, they can excise into a lytic cycle at some other time and location to infect new cells in a new environment to propagate themselves. Lytic phage cannot do this

Two lytic phage with moderate sequence homology infect an E. coli culture. Upon examination of the lysate theoriginal two phage are found along with a new phage that shows characteristics of the original two phage. Provide a two word molecular/genetic explanation for how this third phage was created. (Exam 2)

They recombined

Phage lambda can lysosgenize into a bacterial genome by site specific recombination. Phage Mu can also lysogenize by ______ into the genome of the host (Practice Exam 2)

Transposition

Following mutagenesis you want to quickly identify mutant genes. What mutagenesis strategy would you use? (Practice Exam 2)

Transposon mutagenesis

Transposon insertions are often flanked by short direct DNA repeats. How does this occur? (Exam 2)

Transposonase causes staggered cuts in target DNA which are then filled-in by DNA polymerase resulting in repeat sequences

Do CRISPR arrays ever have more than 20 spacers? (Practice Exam 2)

Yes

Has any group ever developed an improved version of Cas9 that recudes off target DNA cleavage? (Practice Exam 2)

Yes

In Barrangou et al, were mor ethan one newly acquired spacers ever found in the CRISPR array? (Practice Exam 2)

Yes

Is it possible to isolate phage mutants that alter their decision or ability to lysogenize? If so, explain how this might be possible. (Problem Set 7)

Yes of course. The ability of phage to lysogenize requires phage genes/proteins. Such gene products include regulatory factors and enzymes to integrate the phage genome

Can a lysogenic phage change the genotype of a bacteria? Explain (Problem Set 7)

Yes, of course. They can do so in two ways: 1) if they integrate into the bacterial genome they may disrupt a gene; 2) the insertion of the phage now adds all of the phage genes to the bacterial genome

This pET28 vector is widely used for protein expression in E. coli. Itcontains two origins of replication. The purpose of the f1 origin is to make ______________. (Exam 2)

single stranded DNA

Salmonella undergoes phase variation to change what flagellin gene is expressed. Phase variation involves a DNA inversion even between the hixR and hixL sites. Draw the product of an inversion event and explain why a different flagellin gene is expressed (Exam 2)

The "hin" region is inverted and consequently, there is no promoter to drive expression of fliB and fliA, the latter which serves as a repressor for fliC. Therefore, fliC is now expressed

In the pET expression system how many promoters does T7 RNA polymerase recognize from the chromosome and how many from the pET plasmid? (Problem Set 7)

0, 1

The estimated number of bacteria killed per second on Earth is: (Exam 2)

10^23

A phage P1 lysate was grown on a pool of random Tn10 insertion mutants (Tet(r)) in an otherwise WT (trpA+) E. coli strain. The pool contained approximately 5,000 independent transposition events. This lysate was then used to transduce a trpA- recipient. Can you explain with a diagram how to isolate a Tn10 insertion linked to the trpA gene in E. coli?

5,000 colonies with independent Tn10 insertions around the genome were pooled (Parent strain is trpA-) and infected with phage P1 (P1 packages ~100 kb of DNA). ~1 in 100 phage are transducing phage of which 1 in 50 will carry typA+ gene. Infect the recipient strain (transduction) and plate on minimal media (no tryptophan) plus Tet. Only colonies that can grow are transductants where the Tn10(Tet(r)) is linked to trpA+

In a phage display library prepared from genomic DNA, what fraction of phage clones would you expect to specifically bind to a particular protein target? (Problem Set 7)

>1/10,000

You have isolated a lambda vir mutant that forms clear plaques on WT E. coli. In E. coli you place a plasmid that constitutively expresses the cI gene at high levels. To your surprise the lambda vir mutant also forms clear plaques on this strain. Can you provide an explanation for how this could occur? (Exam 2)

A cis operator mutation(s) occurred in lambda vir such that cI can no longer bind and repress gene expression

You have isolated a lambda vir mutant that forms clear plaques on WT E. coli. In E. coli you place a plasmid that constitutively expresses the cI gene at high levels. To your surprise the lambda vir mutant also forms clear plaques on this strain. Can you provide an explanation for how this could occur? (Practice Exam 2)

A cis operator mutation(s) occurred in lambda vir such that cI can no longer bind and repress gene expression

Briefly, what does "lysogenic conversion" mean? (Exam 2)

A commensal bacteria is lysogenized by a phage that carries virulence genes and therefore converting that bacterium into a pathogen

Briefly explain how phage can become resistant to CRISPR acquired immunity (Practice Exam 2)

A phage could develop a mutation in the protospacer sequence suc that the CRISPR spacer no longer recognizes it for cleavage

Your text describes Mycobacterium phage genomes as being mosaic. Descibe how this occurs in nature, i.e. how are mosaic phage created? (Problem Set 5)

A single Mycobacterium cell is infected by two or more phage. These phage replicate their genomes, generating ~hundred genomes per phage per cell. If the genomes hare homologous regions they ill recombine creating chimeric phage. Non-homologous recombination between phage can also occur. Given that ~10^23 bacteria are infected by phage per second, all sorts of different combinations or mosaic phage can be created and amplified (as long as they are fit)

Below is a diagram of an engineered transposon that includes a promoterless lacZ gene. Assuming the transposon inserts into an ORF, how often would it be translation fused to that protein? (Practice Exam 2)

About 1/6 insertions would lead to a translation fusion

The rise of antibiotic resistant bacterial pathogens is a major global problem. List two mechanisms that allow antibiotic resistant genes to be transferred in bacterial populations (Exam 2)

Antibiotic resistant genes can be spread by phage (transduction), conjugative elements and natural transformation. Also, transposons can carry these genes facilitating their rapid spread in populations

A phage P1 lysate was grown on a pool of random Tn5 insertion mutants in an otherwise WT (hisA+) E. coli strain. The pool contained approximately 10,000 independent transposition insertions. To isolate a Tn5 insertion linked to the hisA gene what type of strain would you transduce and what selective media would you use? (Practice Exam 2)

As a recipient use a hisA- mutant. For selective media use minimal media without histidine and include kanamycin. The only colonies that should grow are transductants with a Tn5-kan linked to hisA+

What is the most abundant (numerous) life-form on Earth? (Exam 2)

Bacteriophage

What is the most abundant life form on this planet? (Practice Exam 2)

Bacteriophage

When M13 phage are secreted from a cell what is the nature of their genome: RNA or DNA; double or single stranded, linear or circle? (Exam 2)

Circular ssDNA

In one sentence descrbied why CRISPR has recently become is a powerful technology that is widely used to edit eukaryotic genomes, i.e. why is it so useful? (Problem Set 5)

CRISPR is a programmable restriction enzyme system that allows researchers to efficiently cut NDA anywhere in the genome of live cells. Thus deletion or insertion mutations can be created anywhere in the genome of nearly any organism

A new dog breed was bred that contained many desirable traits.However, unfortunately, this breed contained a viral derived oncogenethat sadly caused cancer at an early age. Using genomics you identifiedthe causative gene and sought to selectively inactivate this gene amongthe dog 2.5 million bp genome. What technology discussed in class could allow you to do this? (Exam 2)

CRISPR with Cas9-gRNA

To edit a mammalian genome what two CRISPR elements are required or commonly used? (Practice Exam 2)

Cas9 enzyme and gRNA

When a lambda prophage excises out of the genome in what configuration is the genome, linear or circular? (Exam 2)

Circular

Draw the general structure of a composite transposon (Exam 2)

Inverted repeats (L to R)- Gene cassette (cargo genes)- Inverted repeats (R to L) (includes transposase gene)

Crick isolated FC0 rII mutants that he found could be suppressed by secondary mutations. What was the nature of the FC0 mutations and how did this suppressor work? (Exam 2)

FC0 mutations were small deletions/ insertions that caused frameshift mutations. The suppressors restored the reading frame by making compensatory insertions/ deletion mutations

True or False: A lawn of bacteria with plaques of different sizes is shown. The large plaques indicatephage with large genomes while small plaques indicate phage with small genomes.

False

True or False: A phage lambda mutant contains a defective N gene but will nevertheless proceed in a normal lytic cycle. (Exam 2)

False

True or False: During a T7 lytic infection all of the phage genes are expressed at the same time? (Exam 2)

False

In phage display, how are particular phage amplified? (Problem Set 5)

From a library of millions of different random clones (phage) high affinity binders (recombinant phage) can be easily identified as binding to the target on a column. Those phage are then eluted (purified) and can be plated on a lawn on bacteria to isolate single plaques (derived from a single phage particle).

In one sentence explain what phage display is used for (Problem Set 5)

From a random library of recombinant phages it allows the isolation of peptide sequences that specifically bind to a target molecule of interest, e.g. a specific protein

You isolated a new bacterial species and its genome was sequenced and found to contain a prophage. Experimentally how could you isolate viable phage from this bacterium? (Exam 2)

Induce DNA damage such as UV treatment, which typically cause prophage to excise and enter a lytic stage. Viable phage particles can then easily be isolated from such treated cultures

In one sentence, explain why the CRISPR/cas9 system is so powerful for editing mammalian genomes (Exam 2)

It acts as a programmable restriction enzyme to cut any gene of interest which can lead to Indels or facilitate recombination of heterologous DNA

What is unusualy about the biology of phage Mu? (Exam 2)

It functions/replicates as a transposon

If a bacteria contains a fully functional CRISPR system what would happen to a bacterium that acquired a space sequence from its own genome? (Practice Exam 2)

It would cleave its own genome and likely die

Phage lambda, 424, and 21 are all lambdoid phage. Lysogens of these phages are indicated by writing the phage name in parenthesis after the bacterial strain name. c. What is unusual about the phage resistance profile of K-12(HK22)? (Problem Set 9)

K-12(HK22) lysogen is resistant/immune to HK22 infection, but surprisingly it is also resistant to phage lambda and 424. This is not expected because lambda and 424 immunity are clearly different than HK22, i.e. the lysogens of the former phage are senstivie to HK22 infection, but are both homo-immune

Why does wild-type phage lambda produce turbid plaques on E. coli? (Exam 2)

Lambda can lysogenize, thus within a plaque there are lysogens growing so the plaque is not clear

Phage lambda, 424, and 21 are all lambdoid phage. Lysogens of these phages are indicated by writing the phage name in parenthesis after the bacterial strain name. a. Why does lambda grow on K-12(424) and K-12(21), but not on K-12(lambda)? (Hint: consider immunity) (Problem Set 9)

Lambda has different immunity (i.e. CI repressor and operator sequences) than phages 424 and 21 and therefore lambda can grow on both of these lysogen strains, but not a lambda lysogen

Phage lambda, 424, and 21 are all lambdoid phage. Lysogens of these phages are indicated by writing the phage name in parenthesis after the bacterial strain name. b. Why does phage HK22 grow on K-12(lambda), K-12 (424) and K-12(21), but not K-12(HK22)? (Problem Set 9)

Lambdoid phage HK22 apparently has a different immunity (i.e. CI repressor and operator sequences) than phages lambda, 424 and 21 and therefore it can grow on these lysogenic strains

Francis Crick used the rII gene from phage T4 to elucidate the genetic code. Here rII mutations were identified as ______ plaqus. Additionally, T4 rII revertants/ suppressors were selected for growth on a ________ (Practice Exam 2)

Large (rapid lysis) E. coli lambda lysogen

When a lysogenic phage turns a commensal bacterium into a pathogen this is called __________. (Exam 2)

Lysogenic conversion

In phage Lambda the cro gene was deleted. Upon infection of an E. colicell what is the most likely outcome? (Problem Set 7)

Lysogeny

Of the phage we discussed, which one does not package its DNA by a "headful" mechansim? (Practice Exam 2)

M13

Of the phage we discussed, which one does not package its DNA by a "headfull" mechanism? (Exam 2)

M13

What phage discussed in class does not package its genome by a "headful" mechanism?

M13

What phage system do molecular biologists use to make single stranded DNA? (Practice Exam 2)

M13

In one sentence describe why M13 cloning vectors are useful for site directed mutagenesis (Problem Set 5)

M13 cloning vectors provide an easy method to generate single stranded DNA to serve as template for DNA synthesis with a mutagenic primer

What phage system do molecular biologists use to make single stranded DNA? (Exam 2)

M13/f1

After the initial injection of Lambda DNA, the _______ protein must be made in order for the cII gene to be expressed. (Problem Set 7)

N antiterminator

A protein is thought to form transmembrane helices. To elucidate membrane topology three phoA translational fusions were made. On XP indicator plates, when PhoA localizes outside the cytoplasm the colonies turn blue (PhoA+) and when PhoA is located in the cytoplasm they form white colonies (PhoA-). Base on the below results draw the membrane topology for the protein describing the location of the 3 domains (1,2 & 3) N-1-PhoA PhoA- N-1-2- PhoA PhoA+ N-1-2-3-PhoA PhoA- (Exam 2)

N-1 and 3-C on inside of membrane 2 outside membrane

Would you expect a lambda lysogen to provide immunity, as discussed in class, to a phage that did not belong to the lambdoid family? (Practice Exam 2)

No

You want to isolate a missense mutation in the dnaB gene. Is transposon mutagenesis a good strategy? Yes or no (Practice Exam 2)

No

A student designed a novel phage genome that contains genes from different phages. Although the gene products work together, this synthetic phage contained the identical promoters to control early, middle and late gene expression. Would the phage be viable? Why? (Exam 2)

No, phage gene expression needs to be temporally control to ensure orderly expression and assembly of phage components

Can the T4 phage genome fit inside the "head" of phage lambda? Explain (Exam 2)

No, the genome is too big

In the pET expression system how many promoters does the T7 RNA polymerase recognize in the cell? (Practice Exam 2)

One, which is plasmid encoded

If a cell contained a plasmid that overexpressed the lambda, cII gene and those cells were infected by lambda, what would happen in terms of the lytic/ lysogeny decision? Explain in molecular terms (Problem Set 9)

Overexpression of cII would favor the lysogeny decision because it would activate cI and int expression resulting in the repression of the strong lambda Pr and Pl promoters and integration into the attB site

You identified a mutant form of a human protein that is an excellent biomarker predictor of cancer. With earlydetection thousands of lives could be saved! What technology discussed in class could you use to specifically detect this biomarker? (Exam 2)

Phage Display

In your research you identified a protein that causes cancer. To study this protein and to develop a possible cure you want to identify a peptide that binds and possibly inhibits this protein. What technology discussed in class can help you? (Exam 2)

Phage display

You have isolated a phage that infects E. coli from the Laramie sewer. Upon careful examination of the plaques you find they are turbid. What type of phage have you isolated? (Practice Exam 2)

Probably a lysogenic phage. However, the identity is unknown, i.e. there are many different types of phage

In the E. coli CRISPR system discussed in class what prupose do Chi sites (DNA sequence) serve? (Practice Exam 2)

Self-recognition

Salmonella uses the Hin invertase to phase vary which flagellin genes are expressed. A genetic engineer has taken the invertase cassette and fused it to a promoterless lacZ gene. When this recombinant Salmonella strain is plated on X-gal (substrate for LacZ enzyme) plates what do you expect to see in terms of blue/white colonies and why? (Practice Exam 2)

Some colonies will be bluea nd some will be white because the hin cassette will invert orientations in the population and only the orientation shown will provide a promoter to drive lacZ expression.

What function does CRISPR RNAs (crRNAs) serve? (Problem Set 5)

They act as the guide or specificity RNA that pairs by complementary with the target DNA that is then cleaved by the Cas, e.g. Cas9, endonuclease

To isolate a Tn5 insertion within a 1 kb distance upstream of the E. coli dnaC gene about how many random hops into the genome would be needed, i.e. what size would your transposon library be? (Exam 2)

The genome is about 4.6 Mbp, so 4,600 hops could theoretically provide an insertion every 1 kb. Insertions are stochastic so the library would need to be larger, e.g. 5,000 to 10,000, to ensure reasonable probability of a linked insertion

What is the most significant advantage transposon mutagenesis offers over random chemical/UV mutagenesis? (Exam 2)

The mutations are tagged and hence they can be rapidly identified

You wish to introduce a transposon into a bacterial cell. List fundamentally distinct ways the transposon DNA can be introduced into a recipient cell (Exam 2)

Transduction, transformation and conjugation

A bacterium developed resistance to a particular phage by the Cas-CRISPR system. However, a phage was isolated that overcame or suppressed this resistance system. HOw does the suppressor likely work? (Exam 2)

The phage can develop a mutation in its target sequence such that the crRNA no longer recognizes the phage DNA

Phage lambda, 424, and 21 are all lambdoid phage. Lysogens of these phages are indicated by writing the phage name in parenthesis after the bacterial strain name. d. Based on your answer in C, can you provide a theoretical explanation for how this might occur? (Problem Set 9)

The simplest explanation is that the HK22 lysogen produces an alternative function that results in resistance to both lambda and 424 but not phage 21. Two common ways a lysogen may do this are to produce a protein that (i) degrades the DNA of certain phages or (ii) modifies their receptors on the cell surface. However, in this case, the HK22 lysogen produces a protein called Nus that prevents antitermination by the N-protein, so the late gene products required for lysis for lambda and 434 cannot be made. (Note: I'm only exprecting a reasonable mechanistic answer, not necessarily the Nus mechanism)

An E. coli cell is engineered to overexpress the CI repressor. This strain is then infected with phage Lambda. What will happen? (Problem Set 7)

The strain will be resistant

What function do inverted repeats (IR) play at the ends of transposons? (Practice Exam 2)

The transponase recognizes the IR sequences and cuts those ends to allow the element to transpose. The IR elements define the boundary of the transpson and everything in between is part of the element

In a bacterial genome why does the Cas9-crRNA complex not cut its cognate spacer sequence in the CRISPR array? (Exam 2)

There is no adjacent PAM sequence

Predict what will happen to the cells by the end of a 2 hour time-lapse movie. Background: Green dots are phage lambda and black rods are E. coli cells. The cells are in rich media and thus can grow. Two cells with one green dot: (Problem Set 8)

These cells are infected with lambda at MOI of 1 and thus will likely enter a lytic life cycle. In so doing they will express the phage reporter pgD-GFP, which will make the cells fluoresce green before they lyse

Predict what will happen to the cells by the end of a 2 hour time-lapse movie. Background: Green dots are phage lambda and black rods are E. coli cells. The cells are in rich media and thus can grow. Cell with three green dots: (Problem Set 8)

This cell is infected at a MOI of 3 which favors the lysogeny pathway. Because of this the cell will likely express sufficient levels of CII to drive the expression of the Pre-mCherry reporter which will make the cell fluoresce red. A lysogenetic cell will not lyse

Predict what will happen to the cells by the end of a 2 hour time-lapse movie. Background: Green dots are phage lambda and black rods are E. coli cells. The cells are in rich media and thus can grow. Cell with no green dot: (Problem Set 8)

This cell is not infected and will thus grow and divide and stay black

Why are molecular biologists concerned about off target effects with CRISPR genome editing? (Practice Exam 2)

This could result in other mutations in the genome, which could result in a diseased individual or an unwanted/ complicating phenotype

Why did Francis Crick use the T4 rII system to elucidate how the genetic code works? In other words, why is this system so powerful? (Problem Set 5)

This is a relatively unique system where there is a good screen for rII mutants (large T4 plaques, i.e. rapid lysis) and a powerful selection for revertants/supressors (plaque formation on an E. coli lambda lysogen)

Bacterial geneticist often use "mini-transposons" where the transponase gene is located outside of the transposable element; often on a suicide vector (i.e. does not replicate in cell). What advantage does this offer over using a wild-type transposon? (Exam 2)

This means the transposon is not a "live" element. Without the transposase gene the transposon cannot hop. Therefore, if the transposonase gene is removed from the cell the mutation will be stable

What biologial purpose does phase variation serve Salmonella? (Exam 2)

To evade the immune system

When thinking about phage, what is their ultimate biological goal? (Problem Set 7)

To successfully reproduce themselves

True or False: An E. coli cell can harbor more than one Lambdoid phage. (Problem Set 7)

True

True or False: During the infection cycle phage the Lambda genome exists in both a liner and circular forms. (Problem Set 7)

True

True or False: In nature two different phage that are partially homologous, can infect one bacterium cell and recombine their genomes to create a new chimera phage. (Problem Set 7)

True

True or False: Is it possible for a bacterium to gain a fitness advantage when a lysogenic phage integrates into its genome? (Problem Set 7)

True

True or False: Phage can be used as antibacterials agent to treat human infections. (Exam 2)

True

You obtained a lambda phage that contains a Tn5 (Kan(r)) transposon in its genome. You infect E. coli with this phage and obtain Kan(r) colonies. When considering where Tn5 resides in these cells list two possible locations (generally speaking) (Practice Exam 2)

Within the lambda lysogen genome or elsewhere in the bacterial chromosome where Tn5 has transposed to a new location

A lambda lysogen can excise and enter the lytic life cycle by the cleavage of the cI repressor. What host factor facilitates cI cleavage? a. Lon protease b. FtsH/ HfIB protease c. RecA bound to ssDNA d. ClpA/B protease e. Proteasome (Practice Exam 2)

c. RecA bound to ssDNA

For the pET T7 expression system discussed in class: a. Where are the T7 RNA polymerase gene located? b. How is the pET system regulated, i.e. how is protein expression turned on? (Exam 2)

a. It is integrated into the chromosome b. Addition of IPTG (lactose) that binds LacI and blocks repressor function

Biological organisms make decisions that govern their behavior. Although lambda does not have a brain it does decide whether to go into lytic or lysogenic program a. What happens if cII levels are high in the cell? b. If a lambda phage had cIII mutation what would happen? c. What two cellular/environmental inputs/signals influence cII levels in the cell? d. What happens when cro levels are high? e. What woudl happen to lambda with respect to lytic/lysogenic growth if the N gene was inactivated? (Exam 2)

a. Lysogeny is favored b. Lytic program c. MOI and growth rate d. Lytic program favored e. Lambda is dead. It cannot enter the lytic or lysogenic programs

Francis Crick used the rII gene from phage T4 to elucidate the genetic code. a. What phenotype allowed him to isolate rII mutations? b. What phenotype allowed him to isolate suppressor/revertant mutations? (Exam 2)

a. Rapid lysis (large plaques) b. T4 growth on E. coli lambda lysogen

At the heart of phage lambda's convoluted mechanism for deciding whether to lysogenize or enter lytic growth is the competition between the cI and cro repressors at the below operator site. In this operator there are 3 binding sites for cro and cI and their relative binding affinites. a. If the sequence of Or3 was changed such the cro binds at reduced affinity what would happen in the battle of repressors and in lambda's decision to lysogenize. b. Similarly, if Or1 was changed so cI bound at reduced affinity what would happen? (Exam 2)

a. This mutation would hinder cro binding and thus favor cI binding resulting in more lysogeny events b. Similarly, a Or1 mutations would block cI binding, favoring cro binding and the decision for lytic growth

How are new "repeats" added to CRISPR arrays? a. Transposition b. Site specific recombination c. Homologous recombination d. Staggered cuts and DNA synthesis e. Mechanism unknown (Practice Exam 2)

d. Staggered cuts and DNA synthesis