MOLB 4440 Microbial Genetics Final Review

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?

The below gene has a mutation and you place a plasmid with the wild-type gene (orfA) with its promoter into that strain but it does not complement. Provide two different reasons for why the plasmid does not complement. orfA * - orfB * mutation at end of orfA (Exam 1)

1. The mutation is dominant 2. Mutation has polar effect on orfB

The mature form of Ribonuclease contains 124 amino acids. Like many bacterial proteins the N-terminal fMet residue is cleaved off to generate the mature protein. How many base pairs does the complete ORF contain? (Problem Set 3)

3 x 126= 378 (124 amino acids plus the start (M) and stop codons; 126 x 3)

In order for DNA polymerase to correct (edit) a mistake during DNA replication what precise activity must that enzyme have? (Practice Exam 1)

3' to 5' exonuclease activity

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

In bacteria what is the physiological significance of the ribosome reading mRNA in the 5' to 3' direction, while RNA polymerase reads DNA in a 3' to 5' direction?

This allows transcription and translation to be coupled, i.e. a ribosome can translate an mRNA while the mRNA is still being transcribed

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

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 bacteria "adapt" without mutations to new environmental conditions? If so, explain how this could occur in general molecular terms. (Problem Set 3)

Yes, at some level all cells adapt to different environments. Adaption can occur by changing gene expression, typically one to many genes in response to an environmental change.

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

During translation the template mRNA is read in which direction? (Practice Exam 1)

5' to 3'

In DNA replication and transcription the newly synthesized strand is being made in which direction? (Practice Exam 1)

5' to 3'

In which direction does the ribosome read the mRNA template? (Exam 1)

5' to 3'

The below DNA samples were treated with DNA polymerase, Klenow fragment. Write the products of the reaction. (Problem Set 1)

5'-AGCCTAGGATCCTAG-3' ————————————> 5'-AGCCTAGGAT-3' Overhang removed by 3' to 5' exonuclease activity 3'-TCGGATCCTA-5' 3'-TCGGATCCTA-5' 5'-AGCCTAG-3' ———————————-> 5'-AGCCTAGCATCGTTAT-3' Overhang filled in by DNA polymerase activity 3'-TCGGATCGTAGCAATA-5'. 3'-TCGGATCGTAGCAATA-5'

Below is a double strand DNA sequence. You have sequence to believe this fragment encodes a promoter, Shine-Delgarno and start codon. Identify those elements (and parts) and comment on spacing. Remember these are two DNA strands and thus two sequences to interrogate. 5'-TAACGGCATCCGGAGGACCTCCTGGGTAAGGCCATTATAGGTACCGGATGGGACCCTGTCAACC-3' 3'-ATTGCCGTAGGCCTCCTGGAGGACCCATTCCGGTAATATCCATGGCCTACCCTGGGACAGTTGG-5' (Problem Set 4)

5'-TAACGGCATCCGGAGGACCTCCTGGGTAAGGCCATTATAGGTACCGGATGGGACCCTGTCAACC-3' 3'-ATTGCCGTAGGCCTCCTGGAGGACCCATTCCGGTAATATCCATGGCCTACCCTGGGACAGTTGG-5' _____ ______________ __ __________ _____________ Start Codon SD Tx start. -10 region -35 region Spacing is good: 17 bp between -10 and -35 and 7bp between SD and start AUG

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+

If a bacterial genome contains 7.0 megabases (Mb) of DNA about how many genes would you expect that organism to contain? (Problem Set 1)

A bit less than 7,000 genes

Give an example for how a mutation can block the production of a gene product (e.g. protein) without changing the DNA sequence of the open reading frame (ORF)? (Problem Set 1)

A mutation could inactivate a promoter or prevent an mRNA from being translated

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)

What is the difference between a transcriptional and a translational gene fusion when used as a reporter? (Problem Set 2)

A transcriptional gene fusion is a fusion of a promoter of interest with a reporter gene (e.g. lacZ), while the latter fuses the promoter and part or all of the ORF of interest with a reporter protein (i.e. in-frame protein fusion).

Label a bacterial gene (Exam 1)

Activator site/operator -> Promoter (includes -35 & -10) -> +1 -> Shine Delgarno -> Start -> orfA -> stop -> Transcription terminator Coding strand= 5' -> 3' and contains all of the things mentioned Template strand= 3' -> 5'

How many copies of each gene (alleles) do Gram-negative bacteria have in their genome? (Problem Set 1)

All bacteria are haploid, so they only have one copy of each gene. Note, there are a few exceptions, e.g. rRNA genes

You isolated a missense mutant in DNA helicase (dnaB1) that does not grow on rich media (fast growth), but does grow on minimal media plates (slow growth). Armed with a powerful selection (no growth on rich media) you identify an extragenic suppressor of dnaB1 that maps to dnaC. The frequency for isolating the suppressor is ~1 in 10^10 (i.e. rare event). Intrigued by this result you test five other dnaC alleles from your freezer collection and none of them suppress dnaB1. What type of suppressor did you isolate? (Exam 1)

An allele specific suppressor

A mutant is isolated that cannot grow on minimal media but can grow on minimal media supplemented with arginine. In genetic terms what is the phenotype of this mutant (be precise)?

Arginine auxotroph

Which of the following amino acid changes can result from a single bias change: A) Tyr -> Val B) Ala-> Pro C) Gly->Ala D) His-> Glu E) Met-> Arg (Problem Set 2)

At least two changes: Tyr-> Val, His-> Glu Single change possible: Ala-> Pro, Gly-> Ala, Met-> Arg

Eukaryotes typically engage in sex to produce offspring. In contrast, bacteria are haploid and do not require sea to produce offspring. In genetic terms, what does this mean about the relationship of bacterial offspring? (Problem Set 1)

Bacteria offsprings are clonemates, meaning they are genetically identical to one another (unless a mutation arose during replication).

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

Bacteriophage

For oriC we discussed cis and trans elements/factors. Explain how they are different (Problem Set 1)

Cis elements are sequences within the DNA itself, e.g. binding sites for proteins. Trans factors are protein that bind to particular DNA sequences

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

The below sequence was found for an internal mRNA fragment isolated from an E. coli cell. Assume this sequence corresponds to the middle of an ORF. Deduce the correct read frame and amino acid sequence of the protein you would expect the fragment to encode. (Problem Set 2)

Chose whichever one makes the longest polypeptide

A fast food chain is considering adding new preservatives to their food so the FDA tested those compounds in Ames tests. Below are the results where two different His auxotroph mutants were tested for reversion on minimal media plates. What specifically can you conclude about compound #1 and #2? Growth on hisA frameshift mutation for compound 1 and not compound 2. No growth on hisA missense mutation for compound 1 and growth for compound 2. (Exam 1)

Compound 1 cause insertion/deletion mutations, while compound two causes base substitutions

In eukaryotic genetics, scientists typically do not observe phenotypes following mutagenesis and screening of progeny. In contrast, in bacteria genetics phenotypes are readily observed following mutagenesis. The reason for this is that eukaryotes are typically _______________ and bacteria are _________. (Exam 1)

Diploid Haploid

What is the major protein that initiates DNA replication in E. coli and where does it bind? (Practice Exam 1)

DnaA and it binds to oriC

If a mutation inactivated mismatch repair in E. coli how many mutations, on average, would you expect to arise from a single round of DNA replication? (Problem Set 1)

E. coli DNA pol III makes one mistake about once per 10,000 bases and the genome is ~4.6 Mb, so ~46 mistakes, but editing corrects 99% of these mistakes, so I would expect on average 0.46 mutations per genome replication

Provide a reasonable estimate and rationale for how many promoters the host RNA polymerase (with all of its sigma factors) recognizes in the E. coli K12 genome. Secondly, provide an estimate and explanation for how many promoters phage T7 RNA polymerase recognizes in the E. coli genome (Problem Set 5)

E. coli has around 4,300 genes and each gene must have a promoter. I would thus estimate a similar number of promoters as genes, with the following caveats, some genes have multiple promoters while other genes share one promoter in an operon. T7 prmoters are specific to phage T7 and thus there are not natural T7 promoters in the E. coli genome. If a pET plasmid is introduced into E. coli then that plasmid would have one

What is the major difference between the two trees of life we discussed in class? (Problem Set 1)

Eukaryotes are now represented as a subclass within archaea

As discussed in class, what is the phenotypic difference between a flagellar mutant and a chemotaxis mutant, i.e. describe the phenotypes for each class of mutants. (Problem Set 1)

Flagellar mutants are "motor" mutants so the bacteria cannot swim/move. Chemotaxis mutants make flagella and swim, but cells cannot direct or "steer" their movements

List two mechanisms that stop transcription? (Exam 1)

Factor-dependent and factor-independent termination

Are some bacteria diploid (contain two sets of each gene that are different)? (Practice Exam 1)

False

Do bacteria always only have one copy of their chromosome in the cell? (Exam 1)

False

In order for a bacteria cells o produce progeny it must undergo meiosis? (Exam 1)

False

Under what conditions does E. coli have multiple copies of its chromosome? (Practice Exam 1)

Fast growth

To the above forward primer engineer in an EcoRI restriction site and to the reverse primer engineer a HindIII restriction site. Note: the restriction sites will be engineered at the 5' ends. Write the entire primer sequences. Forward: 5'-ACCTGGCTATATTCCG-3' Reverse: 5'-AGCTGGGTCTATCTAT-3 (Problem Set 1)

Forward: 5'-(GAATTC)ACCTGGCTATATTCCG-3' Reverse: 5'-(AAGCTT)AGCTGGGTCTATCTAT-3

Below is a single strand DNA sequence that you would like to PCR amplify. For each end precisely design a forward and reverse primer that is 16 residues long. Your template DNA is double stranded. 5'-ACCTGGCTATATTCCGGCCGATAGCCTAACCGGCCTCTCTCTCGGAAGGCCTTTAAGAGAGATAGATAGACCCAGCT-3' (Problem Set 1)

Forward: 5'-ACCTGGCTATATTCCG-3' Reverse: 5'-AGCTGGGTCTATCTAT-3'

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

What phenotype would you expect at a higher mutation rate, streptomycin resistance or His- auxotrophy? Why? (Hint: two factors to consider) (Problem Set 3)

His- would have a much higher mutation rate because there are multiple genes that can be mutated and many mutations in any one gene can lead to a null phenotype. For streptomycin resistance only one gene, that encodes S12, can be mutated and there only a few mutation that will result in resistance. The gene is essential.

Which phenotype would you expect at a higher mutation rate, streptomycin resistance or His- auxotrophy? Why? (Hint: two factors to consider) (Exam 1)

His- would have a much higher mutation rate because there are multiple genes that can be mutated and may mutations in any one gene can lead to a null phenotype. For streptomycin resistance only gene, that encodes S12, can be mutated and there only a few mutations that will result in resistance. The gene is essential.

In "Salvation of Doug" the workers represent ________ and cars represent ______ (Practice Exam 1)

Genes cells

In the "Salvation of Doug" how does the geneticist create a "mutation"? (Exam 1)

He ties the workers hands behind their back

An E. coli strain has the following genotype: his- strR dnaAts galK-. List the phenotypes of this strain? (Problem Set 3)

Histidine auxotroph; streptomycin resistant; temperature sensitive for initiation of DNA replication by DnaA mutation; galactose auxotroph

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

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

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

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

You have isolated an internal fragment to a bacterial mRNA. Use Table 2.1 to conceptually translate the mRNA an identify the most probable peptide fragment (Exam 1)

Make the longest polypeptide chain

If the translational start codon in a mRNA is GTG what is the corresponding first amino acid in the protein? (Problem Set 2)

Met

What amino acid(s) has the fewest codon(s) and what amino acid(s) has the most codons? Based on this information which amino acid(s) do you think are more prevalent in protein sequences and why? (Exam 1)

Met and Trp only have one codon. Leu, Ser and Arg have 6 codons each. Based on this I would expect that Met and Trp are rare amino acids in proteins, while Leu, Ser and Arg are common

Below is a figure for the rII operon from phage T4. The promoter is indicated by an arrow and the black tick marks show the start and stop codons for the two genes. The rIIB gene contains regions that are nonessential and essential for protein function. One mutant lacks rIIA function at 42degrees C, but is WT at 30degrees C. For this phenotype, which mutation would you predict to have this property and why? (Problem Set 4)

Mutant 1 would most likely have this phenotype because the mutagen causes base substitutions and not frameshift mutations (mutant 2). A 'classical' or typical Ts mutant phenotype is caused by a mutant protein that is less stable at high temperature because of an amino acid substitution.

Four mutations were mapped to the below operon. Complementation tests found mutations 1&2; 3&4; 1&3 all complemented each other, while mutations 1&4 and 2&3 did not complement. Mutation 1 was mapped to orfA. Identify the genes the other mutations likely map to (Exam 1)

Mutations 1 and 4 map to orfA and mutations map to orfB

Below is a figure for the rII operon from phage T4. The promoter is indicated by an arrow and the black tick marks show the start and stop codons for the two genes. The rIIB gene contains regions that are nonessential and essential for protein function. Mutation 7 produces a functional protein, while mutation 8 produces a nonfunctional protein. Provide a reasonable explanation given the nature of the mutagen. (Problem Set 4)

Mutations 7 and 8 were induced by ICR-191, which causes frameshift mutations. Since mutation 7 is functional the reading from must have been restored, e.g. deletion of 3 bases either next to each other or within a short region of the gene. Although mutation 8 is localized near the 3' end of the gene it likely causes a frameshift mutation that inactivated the protein.

Does RNA polymerase require a primer to initiate synthesis? (Exam 1)

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

Two-factor P1 transductions were done to map several genes. Dra the contransduction frequencies (linkage) for fadL, purF, and aroC fadLpurC (fadL-)= 15% fadLaroC (fadL-)= 44% aroCpurF (purF-)= 63% (Exam 1)

PurF-> aroC (63%) aroC-> fadL (44%) purF-> fadL (15%)

In order for a circular DNA molecular to replicate in a cell it must contain what? (Exam 1)

Ori of DNA replication

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

For the following questions use precise terminology. When two genes within a genome share significant sequence similarity they are said to be __________. When two genes within different genomes share significant sequence similarities they are said to be __________. (Problem Set 2)

Paralogs Orthologs

E. coli DNA polymerase makes about 46 mistakes per genome replication. However, a mutation will arise only 1 per 2,000 generations. What two mechanisms make this possible? (Exam 1)

Proof reading by DNA polymerase and mismatch repair

Diagram the product of homologous recombination between the below repeat elements. Consider gene order. Repeat- A- B- C- Repeat (Exam 1)

Repeat- C-B-A- Repeat

A 4 base pair (bp) insertion mutation occurred in a promoter sequence such that a transcriptional activator still bound the DNA, but transcription was no longer activated. Provide a molecular explanation for how this mutation may work

Such an insertion mutation would turn the DNA helix about 180 degrees, which may prevent the activator from interacting with RNA polymerase

List four differences between phage T4 and phage M13 (Problem Set 5)

T4= dsDNA, large (~180kb), linear, lytic, myoviridae M13= ssDNA, small (~10kb), circular, non-lytic (sheds particles from live cells), Inoviridae (filamentous)

You construct a hisB(deletion) mutant that removed the entire gene. A fellow student plated your mutant, 10^9 cells, on a minimal media plate and noticed that a few colonies grew 3 days later and called them revertants. What is your response? (Exam 1)

The colonies cannot be revertants because the hisB gene was deleted. They must be suppressors

Below is a figure for the rII operon from phage T4. The promoter is indicated by an arrow and the black tick marks show the start and stop codons for the two genes. The rIIB gene contains regions that are nonessential and essential for protein function. Provide a hypothesis for why 5 produces a functional RIIB protein, while the small 4 deletion is non-functional. (Problem Set 4)

The larger 5 deletion mutation fuses the two ORFs in-frame resulting in a functional RIIB protein. In contrast the shorter 4 mutation must fuse the ORFs out-of-frame.

Below is a figure for the rII operon from phage T4. The promoter is indicated by an arrow and the black tick marks show the start and stop codons for the two genes. The rIIB gene contains regions that are nonessential and essential for protein function. Mutation 6 results in a non-functional RIIB protein. PRovide a likely molecular explanation. (Problem Set 4)

The mutations resides in a nonessential region of the protein; therefore a missense mutation is not likely. Instead I would predict mutation 6 causes a nonsense mutation.

When the cell machinery senses a mismatched base pair in the DNA (error during replication), how does the E. coli cell know which strand has the correct sequence? (Practice Exam 1)

The old strand is methylated

What would happen to the PCR reaction if you engineered the above restriction sites and the 3' end? Why? (Problem Set 1)

The oligopoly would not work as primers to initiate DNA synthesis because the 3' ends would be mismatched with the DNA template. Without proper pairing at the 3' end, i.e. the 'business end', DNA polymerase would not have usable primers

What happens if an E. coli ribosome translates an mRNA that has been cleaved prematurely and consequently has no stop codons? (Exam 1)

The ribosome will stall when it reaches the end of the RNA template without encountering a stop codon. A 'tmRNA' can then enter the ribosome as serve as a 'mRNA template' and 'tRNA' to adde a proteolytic tag and stop codon. The nascent protein will be released from the ribosome and degraded

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)

Below is a genetic map. A deletion mutation was found to span the terminator and promoter. Describe in molecular terms what you would expect to happen. Promoter-dnaK-dnaJ-terminator-promoter-hisA (Problem Set 3)

The transcription terminator for dnaKJ operon is deleted as is the promoter for hisA. I would expect the expression of hisA would depend on the dnaKJ promoter.

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

Below is a figure for the rII operon from phage T4. The promoter is indicated by an arrow and the black tick marks show the start and stop codons for the two genes. The rIIB gene contains regions that are nonessential and essential for protein function. Deletion 5 produces a function rIIB gene product, but the gene product for the 4 mutation is non-function. In molecular terms explain what happens to the mRNA and proteins in these mutants (think central dogma). (Problem Set 4)

These deletions remove part of the 3' region of rIIA and part of the 5' region of rIIB and the entire inter genie region. Therefore the rIIA and rIIB genes are fused at the mRNA level and translation of these mRNAs would only result in a single protein, i.e. the downstream rIIB fragment would not be translated independently because there is no SD-start codon.

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

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 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)

What purpose does the Shine-Delgarno (SD) sequence serve? (Exam 1)

This sequence in the mRNA, in conjunctions with a properly spaced start codon, instructs the ribosome where to initiate translation

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

To successfully reproduce themselves

What are the three methods used to introduce DNA into a bacterial cell? (Exam 1)

Transduction, conjugation and transformation

You used ICR-191 to generate a frameshift mutation in the argC gene. Next, you sought to isolate argC+ revertants. What type of mutagen would you use and why? (Exam 1)

Use ICR-191 to create frameshift (small insertions/deletions) mutations. Most of the revertants would likely be intragenic suppressors that restore the reading frame

You seek to engineer a His-tag into the C-terminus of the below ORF by PCR. The tag will be constructed by primer design and PCR amplified and cloned into an expression vector. The forward primer has been designed by you need to design the reverse (R) primer. In this primer engineer six His codons (CAU and CAC) before the stop codon. The 3' end of your primer should have 18 residues that precisely anneal to the given template DNA. In addition, add an EcoRI restriction site to the 5' end of the prier. Last, make sure you properly engineer the stop codon (TAA) into your primer sequence EcoRI site: 5'-GAATTC-3'; 3'-CTTAAG-5' 5'-GGGCTTATCGTCAACGTGTAA-3' (Exam 1)

Your reverse primer: EcoRI site-> stop -> His tag (6 times) (ATG x 6) and then the sequence complementary 5'-CACGTTGAGCATAAGCCC-3'

Three bacteria strains defective in tryptophan synthesis are streaked on minimal medium plates lacking tryptophan and grown at three different temperatures. After incubation for two days the plates appear as shown below. Note: Once tryptophan has been synthesized it can no longer diffuse out of the cell, but in contrast intermediates can diffuse out of the cell. 25 degrees: 1=growth, 2= little, 3= none 33 degrees: 1=growth, 2=none, 3=growth 41 degrees: 1= little, 2= none, 3=growth a. Briefly state the phenotype of the mutants with respect to temperature b. In the pathway below label the arros to show the enzymatic step blocked in each mutant W -> X -> Y -> tryptophan (Exam 1)

a. Mutant 1: Ts- for Typ biosynthesis at 41 degrees Mutant 2: Typ- auxotroph at all temperatures Mutant 3: Cold-sensitive (Cs-) for Typ biosynthesis at 25 degrees b. W-> X (1) X -> Y (2) Y -> tryptophan (3)

You identify two protein sequences in a genome that share 78% amino acid identity across their entire lengths. a. What is the precise bioinformatic term to describe their relationship? b. Do these proteins necessarily have identical functions? Yes or no (Exam 1)

a. Paralogs b. No

A cross is done by conjugation between a trp+ gly+ ser+ rif(s) Hfr donor and a trp-gly-ser-rif (r) F-recipient, selecting recombinants on minimal plates containing rifampicin, glycine and serine. a. Which marker(s) in the cross are being selected? Which are unselected? b. How would you score inheritance (or transfer) of the unselected markers? c. How could you select for Ser+ recombinants, i.e. instead of Trp+ recombinants? Your petri plate would contain:(Exam 1)

a. Trp+ and rif(r) are selected markers; gly and ser are unselected b. Replica plate recombinants on minimal media plates containing either glycine or serine and test for growth c. Use minimal media plates containing rifampicin, glycine and tryptphan

An average bacterial gene contains how many bps? (Practice Exam 1)

~1,100 bp

What is the upper limit of total genes found in some bacterial genomes? (Practice Exam 1)

~10,000

An average size protein contains about how many amino acids? (Exam 1)

~330 amino acids

The E. coli genome contains about how many genes? (Exam 1)

~4,500

What is the minimum number of genes, approximately, found in a bacterial species genome? (Practice Exam 1)

~500