Genomics final exam

Sustained in situ protein production
and release in the mammalian gut
by an engineered bacteriophage:
What is the point of this experiment

To use phage infected resident gut bacteria to deliver therapeutic agents into the GI tract without having to take daily medication

Hypothesis of the experiment

the lytic phage T4 could help release the GFP from inside the bacteria into the surrounding environment by causing the bacteria to burst open (lyse)

Rationale of the experiment

By using the phage to lyse the bacteria, they could potentially deliver therapeutic proteins (like GFP) produced inside bacteria, to the gut more effectively

Experimental: With phage

significantly increases GFP fluorescence in the supernatant over time, indicating effective release of GFP due to phage-induced lysis, while GFP fluorescence in the cell pellet decreases as the bacteria lyse

Control: Without phage

GFP fluorescence remains high in the cell pellet and low in the supernatant, indicating that GFP is contained within the bacterial cells without phage-induced lysis

AU/OD is arbitrary units/optical density. Why did they divide AU by OD?

the data reflect the amount of protein released per cell, rather than being influenced by the total number of cells

Transcription from phage promoters occurs in waves, first “early” promoters are turned on, then “middle”, then “late” promoters.

What class/function of genes are commonly transcribed at each stage?

Early promoters transcribe genes for DNA replication and host modification, middle promoters for DNA replication and recombination, and late promoters for structural proteins and lysis enzymes

How did they determine which promoter should encode the inserted foreign gene in the phage?

They tried promoters in the early, middle, and late stage of phage reproduction and chose the one with the maximum gene expression but lowest phage propagation impact

Given the chosen parameters (maximal sfGFP levels and minimal impact on phage titer) which promoter would you use and why?

gp22 is an early promoter that enables high expression of sfGFP while minimizing the effect on phage propagation.

Conclusion of experiment 1

Engineered T4 phage can orchestrate the production and release of heterologous proteins in the mammalian gut during the phage infection process, leveraging its co-existence with its bacterial host for a sustained in situ effect

Chimeric infective particles expand species boundaries in phage inducible chromosomal island mobilization

capsid-forming PICIs (cf-PICIs) only require what from helper phages

They only need tail proteins because they encode their own capsid

What is the main function of phage tail proteins

mediate the attachment and infection of host cells, facilitating the transfer of genetic material during the infection process.

SaPI vs cf-PICIs
Which are only found in S. aureus and have very narrow host genera?

SaPIs

SaPI vs cf-PICIs
Which are highly abundant and have a diverse group of hosts across species and genera?

cf-PICIs

cf-PICIs are highly abundant, and
often identical in sequence, across different
species and genera. What does this mean

suggest that these elements are highly efficient at horizontal gene transfer, allowing them to spread virulence and antibiotic resistance genes widely and rapidly

Tail-less cf-PICIs were isolated from the supernatant of induced Kp DSM30104. In addition, supernatants were isolated from induced E. coli isolates containing capsid mutant prophage (either HK106 or HK022, which have different tails). Why capsid mutants?

To make sure the prophage can only donate a tail

What was the purpose of experiment 2

To see if tail from one species specific phage will impact the ability of a different prophage to infect its host.

What was the purpose of experiment 3:
Natural inter-species cf-PICI transfer

transfer of the cf-PICI can occur naturally in mixed bacterial populations, but it requires a compatible helper phage (like HK022) to provide the necessary tails for the cf-PICI capsids

You hypothesize that the gene encoding a therapeutic protein could be encoded into a lytic phage so that it is co-expressed with phage genes during the infection of a patient’s resident gut bacterium, and that phage–bacterial co-existence would lead to the sustained production of your protein.  Which of the options below would be best if the goal is to treat your patient?

High concentrations of your protein and of phage particles are released from each lysed bacterial cell.

Positive-sense (+) viral RNA

is like mRNA and can be immediately translated by host ribosomes

Negative-sense (-) viral RNA

is complementary to mRNA and must be converted
to (+) RNA before it can be translated

Baltimore groups 1 and 2 DNA viruses require

RNA polymerase to transcribe genome and a DNA polymerase to replicate genome

Baltimore group 7 DNA viruses require

RNA polymerase to transcribe genome AND a virus encoded RT to replicate genome

Large DNA viruses vs small DNA viruses:
Which ones DO NOT encode their own polymerases?

Small DNA viruses do not encode their own polymerases.

Baltimore group 6 and 7 (DNA) viruses require

a virus-encoded reverse transcriptase (RT) to replicate their genomes,

Baltimore groups 3, 4, and 5 RNA viruses require:

Virus-encoded RdRp (an RNA-dependent RNA polymerase) to both transcribe and replicate their genome

When is RdRp required following infection by a virus with a (+) ssRNA genome?

RdRp is not packaged within the virion. Instead, the viral genome is directly translated by the host cell's machinery to produce RdRp after infection

When is RdRp required following infection by a virus with a (-) ssRNA genome?

RdRp must be packaged within the virion because the host cell cannot directly translate the (-) ssRNA genome. The RdRp transcribes the (-) ssRNA into (+) ssRNA, which can then be translated into viral proteins and serve as a template for genome replication

Quasispecies

Virus populations are not identical, even within a single host, rather they exist as dynamic distributions of nonidentical but related replicons

How do latent herpesviruses persist in the host cell nucleus without being detected?

combination of histone modifications and regulatory RNAs inhibit viral transcription to limit expression of viral proteins

Marek's disease virus (MDV)

Herpes virus that infects chickens

How would you interpret the cell culture data below?

Disrupting MDV DNA pol exonuclease activity reduces plaque size so MDV shows dependency on DNAP proofreading for effective infection.

provirus

dsDNA moves into the nucleus and inserts into the host genome

Quasispecies in regard to HIV

It takes 5-10 years to develop AIDS, during which time
HIV evolves into patient-specific quasispecies

Early stage (R5; Macrophage trophic) HIV virions

can infect dendritic cells and macrophage that display
CD4 (with CCr5 as co-receptor), and T cells that
display the same

Evolved late stage (X4; T-cell trophic) HIV

initiate infection using CD4 with CXCr4 as a co-receptor (only T cells)

What happens when viruses replicate above
their error threshold?
(1) The population contains too many mutations
and results in dead viruses.
(2) The population does not contain mutations
and results in live viruses.
(3) The population contains too many mutations
and produces more pathogenic viruses.
(4) The population cannot undergo reassortment
and produces less pathogenic viruses.
(5) The population does not contain mutations
and produces more pathogenic viruses

The population contains too many mutations
and results in dead viruses

The table shows HIV genotyping data
from ten patients. HIV in the five
patients previously treated with
tenofovir have become resistant to this
base analogue inhibitor of RT. HIV in
the five new patients (untreated) are
sensitive to tenofovir. What is the RT
amino acid change that is associated
with the development of resistance?

Which of the following describes an activity provided by reverse transcriptase?

(A) It has protein-dependent RNA polymerase activity

(B) It has DNA-dependent RNA polymerase activity

(C) It has RNase H activity that hydrolyzes the RNA strand in an RNA/DNA hybrid

(D) It converts select proteins into their specific mRNA sequence

(E) It has RNA-dependent RNA polymerase activity

It has RNase H activity that hydrolyzes the RNA strand in an RNA/DNA hybrid

Which 3 functions does RT have

RNA-dep DNA pol
DNA-dep DNA pol
RNAse H activity

Your boss gives you $10 million to make Kirkland insulin. Q1: How would you proceed?
(1) Party like it’s 1922 (i.e., extract bovine insulin from pancreases)
(2) Party like it’s 1995 (i.e., clone Babe [the pig from the movie] and isolate his insulin)
(3) Party like it’s 2025 (i.e., just stand in the corner looking at your phone)
(4) Party like it’s 1978 (i.e., clone the insulin ORF into E. coli and overexpress/purify)
(5) Make the hotdogs from 100% bovine pancreases (....probably already are anyway)

Party like it’s 1978 (i.e., clone the insulin ORF into E. coli and overexpress/purify)

Q2: Which of the below is NOT something you need to worry about with regards to using E. coli
to express insulin?
(1) improper protein folding in E. coli
(2) loss of the expression plasmid from the E. coli cell
(3) intracellular accumulation of expressed proteins as inclusion bodies in E .coli
(4) alternative codon decoding rules between humans and E. coli
(5) lack of appropriate post-translational modifications in E. coli

alternative codon decoding rules between humans and E. coli

Plasmid vector component: MCS

sequence containing multiple unique restriction enzyme sites for easy foreign DNA insertion

Plasmid vector component: T7 promoter

drives high-level expression of a gene of interest in systems using T7 RNA polymerase, commonly used in protein production

Plasmid vector component: HIS-Tag

histidine residues added to proteins for purification purposes using metal affinity chromatography.

Q5: How could we test this is the
seq of the correctly spliced gene?

use primer 5’-ctggttcaagggcttta-3’
in conjunction with reverse
transcriptase to make a cDNA copy of
the mRNA, clone it, and then
sequence it

Q7: What do you need to
check before committing
to using NcoI and XhoI?
(1) that these enzymes do
not inhibit reverse
transcriptase activity
(2) that NcoI only cuts
phosphorylated DNA
(3) that a XhoI site is
present within the
antibiotic resistance gene
of the plasmid chosen for
overexpression
(4) that these enzymes do
not cut internal of your
gene of interest
(5) that these enzymes
function on DNA and not
RNA

That these enzymes do
not cut internal of your
gene of interest

A potential issue with producing a human protein in E. coli
involves the codons used

Human genes often use codons that are less frequently used or recognized by E. coli, leading to inefficient translation and lower protein expression

we’ve mentioned silent (aka. synonymous)
mutations a few times in this course. Q9: Do silent mutations
have any phenotype?

Maybe, while they change a gene codon to one that still
encodes the same amino acid, not all codons are equal and
hence there may be a change in phenotype

What could you do to boost insulin production in E. coli?

synthesize a codon-optimized version of the insulin gene

You add isopropyl β-D-1-thiogalactopyranoside (IPTG) to 0.5 mM. Q12: Group E: What is this and why do we add it?

molecular analog of allolactose used to induce gene expression in strains with the DE3 prophage, leading to the activation of T7 RNA polymerase and subsequent transcription of the insulin gene

Why do we run samples from the different steps (U, I, P, S, Ni-NTA, SEC) on the gel rather than just the SEC sample since that is the important one?

ensures each stage of the purification process is working correctly and helps identify any issues

When attempting to overexpress and purify a protein in E. coli it is common to use an IPTG-based expression system, where the gene of interest is only transcribed following addition of IPTG to the culture.  Why is the gene of interest not significantly expressed until IPTG addition?

(A)  IPTG inhibits the phage lysin protein present within pLysS; this prevents cell lysis and therefore enables the expression of the protein of interest

(B)  IPTG binds to and inhibits the activity of LacI which results in the transcription of the T7 polymerase gene, which in turn results in the transcription of the gene of interest which is located downstream of a T7 promoter

(C)  IPTG enables the E. coli to utilize sucrose as an energy source; the greater energy input results in enhanced protein production

(D)  IPTG binds to lacZ which promotes the ability of the encoded protein, LacZ, to bind to the LacO region of the gene of interest’s promoter, enhancing transcription

(E)  IPTG promotes the activity of the T4 DNA polymerase; resulting in enhanced transcription and translation of the gene of interest

IPTG binds to and inhibits the activity of LacI which results in the transcription of the T7 polymerase gene, which in turn results in the transcription of the gene of interest which is located downstream of a T7 promoter

Protozoa: Amoeba example

Brain eating amoeba called N. fowleri

Does N. fowleri have rRNA operons

No, each N. fowleri cell has ~4,000 copies of a 14 kb circular plasmid that harbor rRNA genes.

Protozoa: Flagellates example

T. brucei which causes African sleeping sickness and transmitted by tsetse fly

VSG switching by T. brucei

variant surface glycoprotein expression to create antigenetic variation for immune evasion

VSG switching by T. brucei pattern

Variants arise with degree of regulation because antibodies act to remove early variants, but by then later variants are already emerging

VSG switching mechanism

altering which 15 BES within the genome is active

recombination or gene conversion-mediated feeding of new
VSG sequence

Variant surface glycoprotein (VSG) switching is the major
driver of antigenic variation in Trypanosoma brucei. One
mechanism of VSG switching is what?
(1) inversion of the bloodstream expression site (BES) promoter
such that it drives transcription of a VSG gene located upstream
in the opposite orientation
(2) the mutation of RNA polymerase such that it recognizes a
different gene promoter sequence, which switches which VSG
gene is transcribed
(3) inducing transcription of a VSG gene located within one of
the densely-packed tandem arrays of VSG genes located within
one of the mini-chromosomes
(4) differential gene expression in response to levels of
extracellular iron
(5) the gene conversion-mediated feeding of new VSG sequence
information into the expressed bloodstream expression site (BES)

the gene conversion-mediated feeding of new VSG sequence
information into the expressed bloodstream expression site (BES)

Plasmodium life cycle: Schizogany

asexual reproduction by
multiple fission, producing merozoites.
The merozoites are what generate the
clinical symptoms of malaria

The first Plasmodium chloroquine-resistant isolate was recovered in 1957. In 2025, how could you determine the molecular basis of resistance?

Metagenomic analysis to compare chloroquine-resistant and -sensitive isolates

What happened when comparing chloroquine-resistant and -sensitive isolates of P. falciparum?

identified a gene on chromosome 7 as the major resistance determinant

Hookworm genomics

shed some light on the remarkable ability of these organisms to
suppress the production of pro-inflammatory cytokines

Female schistosoma

heterogametic sex with ZW chromosome

Male schistosoma

Homogametic with ZZ pair

Analysis of male and female sex chromosomes in Schistosoma

suggests that there are distinct regions on the Z chromosome that differ between males and females, which could be indicative of sex-specific genetic variations or differences in chromosome structure and gene content

Tapeworm genome vs Flatworm distant relative

Although gene order has been lost, ancient chromosomal synteny is preserved among these parasitic flatworms

Genome data has been generated for several
species of hookworm. One insight to come
out of this data is what?
(1) insight into the mechanisms by which these
parasites can suppress production of pro-
inflammatory molecules
(2) observation that these parasites do not
express proteins on their surface, and hence no
vaccine can be developed
(3) observation that the rRNA operons of these
parasites are located on plasmids not as part of
one or more chromosomes
(4) that different hookworm species are
unrelated to each other, sharing as much
similarity to C. elegans as they do with each other
(5) that sequencing the genomes of these
multicellular eukaryotes is easy

insight into the mechanisms by which these
parasites can suppress production of pro-
inflammatory molecules

S. cerevisiae mating pheromones

a-factor
α-factor

S. cerevisiae: a cells

respond to α-factor by growing a projection towards
the source of α-factor

Gene organization in the MAT, HML, and HMR loci on S. cerevisiae
chromosome III

MAT locus contains expressed mating allele, but HML and HMR contain silenced copies of a and α so that mating type switching can occur

MAT locus

region on chromosome III responsible for the expressed mating allele in S. cerevisiae.

HML locus

region on chromosome III containing silenced copies of silenced copy of MATα in S. cerevisiae.

HMR locus

region on chromosome III containing silenced copy of MATa in S. cerevisiae.

Which of the S. cerevisiae strain pairs listed below will result
in shmoo formation toward each other?
(1) two diploid cells (one which is a/α, and the other which is α/a)
(2) a haploid “a” cell that produces a-factor and expresses a
receptor that recognizes a-factor, and a haploid “α” cell that
produces α-factor and expresses a receptor that recognizes α-
factor
(3) a haploid “a” cell that produces a-factor and expresses a
receptor that recognizes α-factor, and a a/α diploid cell
(4) a haploid “a” cell that produces a-factor and expresses a
receptor that recognizes α-factor, and a haploid “α” cell that
produces α-factor and expresses a receptor that recognizes a-
factor
(5) a haploid “a” cell that produces α-factor and expresses a
receptor that recognizes α-factor, and a haploid “α” cell that
produces a-factor and expresses a receptor that recognizes a-
factor

haploid “a” cell that produces a-factor and expresses a
receptor that recognizes α-factor, and a haploid “α” cell that
produces α-factor and expresses a receptor that recognizes a-
factor

Gal4 bait and prey technique is used to determine

If a protein interacts with 2 test proteins

Gal4 bait and prey overview

create a bait construct with the Gal4 binding domain fused to your protein and two prey constructs with the Gal4 activation domain fused to each test protein; co-transform these into S. cerevisiae with a reporter gene, and if the bait and prey proteins interact, the reporter gene is transcribed

In the gal4 technique, what do you fuse the “favorite protein” to

The gal4 binding domain

In the gal4 technique, what do you fuse the non-favorite protein to

the Gal4 activation domain

In the gal4 technique, if the bait and prey interact, then what happens

the reporter gene is transcribed.

In the gal4 technique, if the bait and prey don’t interact, then what happens

the reporter gene is not transcribed.

How come most organisms have only 45unique tRNAs

the 3rd base pairs by wobble rules so that fewer codons are needed

Wobble rules

Inosine in the anticodon can pair with uracil (U), cytosine (C), or adenine (A) in the codon, allowing for flexible base pairing

G-U

Decoding CUN in S. cerevisiae

only uses two tRNAs to translate the four CUN codons
(each of which translates two codons)

Candida CUN decoding overall

Candida species translate CUG codons as serine instead of leucine

Candida CUN decoding mechanism

dedicated tRNA_CAG^Ser for CUG codons and a single tRNA _IAG^Leu for CUA, CUC, and CUU codon

Mycorrhiza

A symbiotic association between fungi and plant roots that enhances nutrient exchange.

endomycorrhizas

occur in ~80% of all plants. The fungal hyphae penetrate the cell walls of plant root cells and invaginate the cell membrane to create a greater contact surface area, promoting nutrient transfer

ectomycorrhizas

occur in ~10% of all plants, particularly “woody” plants (e.g., trees). The hyphae of these fungi do not penetrate individual cells within the plant root, rather they make a sheath around roots

Candida species use only two different tRNAs to
decode all four CUN codons (where N is any
nucleotide). How is this possible?
(1) the third base of an anticodon can pair by the
wobble rules (i.e., G-U, I-U, I-A, I-C)
(2) the third base of a codon shows no specificity of
binding
(3) the second base of an anticodon can pair
simultaneously with the second and third bases of a
codon
(4) the third base of an anticodon can pair by the
womble rules (i.e., G-A, I-U, I-A, I-G)
(5) the third base of an anticodon can pair by the
wobble rules (i.e., G-C, I-U, I-A, I-C)

the third base of an anticodon can pair by the
wobble rules (i.e., G-U, I-U, I-A, I-C)

Do all beer yeast make the same metabolites

NO, different strains of beer yeast produce varying metabolites, affecting flavor, aroma, and alcohol content.

Why do yeast strains differ in their alcohol tolerance

allows yeast to survive and continue fermenting in environments with high alcohol concentrations, which is crucial for producing alcoholic beverages and biofuels

Diversity of sporulation efficiency in
industrial S. cerevisiae strains

Beer yeasts have lost their ability to effectively sporulate because they are only exposed to nutrient rich environments

Candida albicans and close relatives harbor a unique barrier to horizontal gene transfer that is not seen in other fungal species.  What is this barrier?

(A)  they have no cell surface glycoproteins and hence lack receptors required for viral infection

(B)  they only grow as spores and hence, due to the thick spore coat, are protected against DNA entry

(C)  they harbor an unusually large number of R-M complexes within the periplasm

(D)  they harbor uracil, not thymine, in their DNA

(E)  they use a non-standard genetic code

E.  They translate CUG codons as serine (while most other organisms on the planet translate as leucine).  Thus, if genes are introduced into C. albicans (let’s say from S. cerevisiae), and these genes have CUG codons in them, these will be translated as serine which may lead to unfunctional proteins

Archaea are most similar to

Eukaryotes with a single membrane and thick cell wall

Archaea are resistant to a wide variety of antibiotics that are primarily produced by

Gram positive bacteria

Why are archaea resistant to gram positive produce antibiotics

these antibiotics primarily act on factors that distinguish
archaea from bacteria

Do archaea have more or less introns than bacteria

More because they are more complex