BioMed Final

Prokaryotic operons are best described as:

A group of genes that is cotranscribed

What is bound to the well in a sandwich ELISA?

Ab

Sandwich ELISA

Ab - Ag - Ab - Ab - Enzyme DAS(double) TAS (triple) specific disadvantage - small Ag may not be able to accommodate multiple Ab to detect specific Ab against specific Ag -detection Ab from sera for TAS -sera Ab bound, Ag captured if Ab present to detect type of Ab (ex: all IgG) DAS works (treat IgG from ser as antigen)

Which trait/traits below are shared by all three types of ELISAs discussed (direct, indirect, sandwich)?

Ab-Ag interactions Detection by observing the enzymatic product of an added substrate

Indirect ELISA

Ag - Ab - Ab - Enzyme 2 ab is conjugated to detection enzyme. larger signal. Covid AB test can be used to detect antigens -use Ab developed against Ag or antibodies -from sera - use immobolized ag to capture sample abs

Direct ELISA

Ag - Ab - Enzyme binds all protein to sample to plate -expensive Direct require antibody to be enzyme linked, dont occur naturally

Which of the following is true for primers for PCR?

Allow specificity of PCR Site for DNA Polymerase to begin synthesis Complementary to target DNA

Assuming that your qPCR reaction is working as-intended, immediately after which of the following steps below would you expect SYBR Green to be fluorescing?

Annealing Extension

Which of the features below from plasmid pUC18 is/are INCORRECTLY paired with its function, if any?

Beta-galactosidase: produces white pigment from X-gal, its inactivation allows for selection of bacteria containing recombinant plasmids

In affinity chromatography, strong protein binding interactions are utilized to facilitate purification. In such cases, ligand for the target protein is affixed to an insoluble support. A mixture containing the target protein is passed over the ligand; the target protein _______, while the remaining proteins __________.

Binds the ligand; flows through the column

You run a western blot on a sample of purified protein and are surprised to find that the entire membrane was detected as your specific protein of interest! What's possible?

Blocking step unsuccessful primary antibody not specific to purified protein

Western blots and ELISAs are similar in that:

Both detect the signal produced by an enzyme conjugated to the Ab

MacConkey agar

Complex (peptone and lactose) Differential and selective

Gene expression (transcription) can be quantified using qPCR by first:

Converting the mRNA into cDNA using Reverse Transcriptase

PCR steps associated with their function

Cycle repetition - allows mass amplification of the target sequence Extension - Maintaining the reaction at optimal temp. for DNA polymerase to add nucleotides Annealing - lowering the reaction temp. so that primers can bind template DNA

What would you expect to happen if the salt was omitted from the binding buffer during DNA purification

DNA would fail to precipitate onto the filter during binding

FRET relies on the idea of spectral overlap between the donor and acceptor fluorophore. In which scenario below is spectral overlap occurring?

Donor emission - 450nm; Acceptor excitation - 450nm

restriction enzyme sites

EcoRV only - yes BamHI - no,

In FRET, emission of the acceptor fluorophore is achieved through excitation of the donor fluorophore. This is possible because the __________ spectra of the donor fluorophore overlaps with the __________ spectra of the acceptor fluorophore.

Emission; Excitation

SYBR Green fluorescence is detected directly after each ___________ step in qPCR.

Extension

Which of the following are typical fusion tags used to protein purification

FLAG-tag GST-tag His-tag

How to determine whether the proteins ZyxA, ZyxB, and ZyxC interact

FRET, Pulldown, Y2H FRET- develop strains w/ each possible set of interactions above being tagged w/ donor and acceptor. (zyxa-b). Pulldowns- tag each proteins (A,B,C) and pulldown any interacting proteins. Y2H: fuse each bait/prey pair to AD/BD of Gal4 protein, visualize interaction via B-gal assay

An advantage of competitive ELISAs is their ability to detect much lower concentrations of the target antigen compared to sandwich ELISAs.

False

Crosslinking is a stand-alone technique to identify interacting proteins.

False

Size exclusion chromatography (SEC) and agarose/acrylamide gels use the same principle - larger molecules will be impeded as they pass through the matrix (gel) or beads (SEC) and will therefore move more slowly.

False

Nickel-NTA columns rely on the affinity of _______ for nickel - proteins containing this motif will bind nickel and therefore "stick" to the column!

Histidine

qPCR data is often analyzed relative to reference or ____________ genes, which are chosen because their expression is relatively stable and provides an accurate "baseline".

Housekeeping

The purpose of a coating (capture) antibody in an ELISA is to:

Immobilize the antigen to a solid support, such as a microtiter well.

Which reporter from the list below was discussed as a screening tool during the cloning lecture?

LacZ/Beta-galactosidase (blue/white)

RT-qPCR (Reverse Transcription qPCR)

More mRNA = more cDNA = more template. mRNA (and therefore gene expression) can be quantified!

Indirect ELISAs have higher sensitivity than direct ELISAs (they can detect lower concentrations of target antigen). This is mostly because:

Multiple secondary antibodies can bind to the primary antibody, amplifying the signal from a single antigen.

how does PCR and gel electrophoresis

PCR - allows you to amplify a gene or piece of DNA gels - allow you to verify that the steps of your cloning experiment are working

In a direct ELISA, the detection enzyme is conjugated directly to the

Primary antibody

You run a western blot on a sample of purified protein and are surprised to find that the entire membrane was detected as your specific protein of interest! Which of the following are possible scenarios (u purified correctly)

Primary antibody not specific to purified protein Transfer step was unsuccessful and no protein was transferred to the membrane Secondary antibody was not able to bind to primary

Which of the following would be the LEAST ideal candidate for a reporter gene?

Protein C - difficult to detect and not present in the host animal, but detrimental to host health

What is the relevance of dNTPS in the PCR reaction?

Provide nucleotides to be added to the newly synthesized DNA

Promoters are responsible for recruiting _____________ to begin transcription. Promoters are typically located ___________ relative to the structural gene.

RNA polymerase; 5'

In order for protein expression to occur (using prokaryotic expression vectors), the gene must be cloned into the expression vector using the correct ___________, meaning that the triplet codons of the gene are in phase with the regulatory sequences present in the vector.

Reading frame

Restriction endonucleases:

Recognize and cleave specific DNA sequences

What is the function of a promoter in prokaryotic expression vectors?

Recruits RNA polymerase

The presence of __________ in the reaction mixture would differentiate a qPCR reaction from a PCR reaction.

SYBR Green

The prokaryotic ribosome binding sequence is also termed the ___________, and is found _____________

Shine-Dalgarno sequence; in the 5' untranslated region

In pulldowns, prey protein(s) is/are able to be pulled down due to their interaction with the _______.

Tagged bait protein

In PCR, melting refers to

The breaking of the hydrogen bonds holding the strands together

During a transcriptional reporter assay, the observable output (e.g. GFP fluorescence, luminescence) is directly correlated to:

The readthrough of the promoter

A melting curve can help to detect contamination, primer dimers, etc. in your qPCR reaction. Melting curves slowly heat the sample, which melts the DNA. This can detect contamination because different sequences will melt at different temperatures based on:

The relative concentration of A-T/C-G basepairs The length of the sequence

How to purify protein

There are 3 ways we could purify ZyxR: size exclusion, ion exchange, or affinity chromatography. Since it has no inherent tags, we can't use affinity -size exclusion -> ion exchange (or vice versa) --> shortcomings: other ~24kDa, negatively charged proteins will copurity --> rectify: check on gel; do add'l ion exchanges w/ slight variations on change clone on his tag. purify via affinity chromatography --> shortcomings: potential contamination by proteins w/ similar tag/affinity --> rectify: check on gel for purify; check for protein fxn to verify tag didnt interfere

Purpose of the washing step in DNA and RNA purification

To remove any lingering contaminants, such as salt or proteins

Double antibody sandwich ELISAs have no direct interaction among the antibodies used, while triple antibody sandwich ELISAs have direct antibody-antibody interactions.

True

In a basic affinity chromatography setup for protein-protein interactions, a "bait" protein is fused to a tag (e.g. GST) and immobilized in a column or on beads. Whole cell lysates are passed over the immobilized "bait" proteins, and bait-binding "prey" proteins are recovered by elution. Those prey proteins are putative interacting partners of the bait protein!

True

Which choice below best describes the typical relative location of the RBS?

Upstream of the coding region and downstream of the promoter

What property of the elution buffer allows the DNA to be resolubilized and collected?

Water in the elution buffer dissociates the sodium ions from the DNA, now polar DNA can return to the solution

Which of the following is NOT a typical fusion tag used in protein purification?

Zyx-tag

where would u insert trasncriptional reporter on the sequence below

after the +1 promoter

where would u insert the translation reporter

after the start codon

Restriction enzymes

an enzyme produced chiefly by certain bacteria, having the property of cleaving DNA molecules at or near a specific sequence of bases. stick ends compatible with similar sticky blunt ends compatible with any blunt

During a DAS or TAS ELISA, the ELISA wells are initially coated with:

antibody

transformation

bacterial uptake of free dna the most used. host could degrade it. linear dna degraded.

A primer that is complementary to itself as well as to the template DNA would

be a poor choice for PCR - primers that are self-compliment are likely to form primer dimers

best represents purpose of the primary antibody in a western blot

binds to the protein of interest and provides a binding site for the successful antibody

Because DNA molecules are all the same ___, gels separate them based on only ___

charged (negative); size and structure

which step in western blot takes place last

detection and analysis

insert-vector ligation

dna ligase , puts vector and insert together

In a reporter assay, a reporter gene is typically fused to a regulatory element (like a promoter). When the promoter is induced for expression, the reporter gene is expressed and can be easily detected. In order for this to occur, the reporter gene must be __________ relative to the promoter.

downstream

SYBR Green is used in the real-time detection of PCR products in qPCR. This is possible because SYBR Green binds to ____________, which is present in the reaction tube in abundance when amplicons are created.

dsDNA

SYBR Green

dsDNA binding dye - detects all dsDNA in sample only fluoresces when bound to dsDNA

During which step is genomic DNA extraction no longer bound to the filter

elution

ELISAs are always used to detect and quantify an antigen, never an antibody.

false

primer design

flanking gene, limited primer site,

good reporter

generate a protein whose activity can be easily detected -be sensitive and should not be expressed endogenously -have broad and linear detection range -not effect the health or physiology of the hst whether it is expressed

lacZ

generates beta-galactasidase (generates blue in presence of X-gal ) pros: no special equipment needed cons: not easy to use in animals

afp

generates green pros: only UV , quantify with fluorometer cons: very stable, once on always on

lux

generates luciferase pro: sensitive , ok models animals cons: reagants expensive,

transcriptional reports

genes used to measure transcription promoter, +1

reporter

genes used to study other genes, conditions

Melt curves for pPCR

gradual inc of temp melts DNA. ssdna doesnt have flourescence, can detect primer dimers if longer melt time etc

Ct values, less abundant or more abundant?

higher Ct value = lower amt of mRNA

What is true about western blot

in successful membrane transfer, all the proteins on the gel are transferred to the membrane

restriction enzyme choice

many in MCS

translation reporters

measure translation of mRNA +1, RBS, codon

purpose of the membrane in western blot

membrane provides a surface for the antibody detection reactions to take place

what terms would you use to describe a bacterium that colonizes in human lungs (37 degrees C)

mesophilic and obligate aerobe

difference between native and denaturing polyacrylamide gels

native gels separate based on charge and size , while denaturing gels separate only on size

no DNA present in elution buffer. why

neglected to add ethanol to binding neglected to add ethanol to wash

purpose of cloning

protein expression sequencing introducing mutations

molecular cloning

purify and insert dna clone vector put vector into host

which technique that we covered could determine whether ZyxR was activating or repressing transcription of zyxABC

qPCR

how to verify cloning worked

selection. antibiotic marker, conveys vector screening confirms insert, by breaking LacZ (produces blue pigment from X-Gal) Plac - induces LacZ with IPTG

during plate counting, what is the name of the process that allows large numbers of bacteria in liquid to be reduced to countable level

serial dilution

Outcome if you attempted PCR with a denaturation temp that was too low

ssDNA would never be formed; the template would remain dsDNA Extension would not occur because DNA polymerase would be unable to bind Primer would be unable to anneal to the temp. strand

stage of bacteria growth between growth and death rate . equilibrium

stationary phase

Purpose of the drying step in DNA and RNA purification

to remove ethanol from the DNA and prevent its interference in downstream applications

conjugation

transfer of bacterial DNA via sex pilus requires specialized conjugation apparatus for donor bacterium

ELISAs are widely used in medical diagnostics, industrial agriculture, and research.

true

Reporter constructs provide an easily measurable output signal to study the transcription or translation of a target gene/operon.

true

Yeast two-hybrid (Y2H) utilizes separated domains of the GAL4 transcriptional activator and a lacZ reporter. In this setup, the separated domains (AD and BD) are incapable of activating transcription unless they are brought in close proximity via the interactions of fused bait and prey proteins.

true

vectors

used to house the insert DNA and deliver to host (plasmids, viruses, cosmids) plasmids: circular DNA, selfreplicating. small.

transduction

virally mediated transfer of DNA to bacteria virus-host specificity

fluorescent detection

•Lower CT = large amount of starting template •Higher CT = small amount of starting template