Week 9 Assignment

Place the steps of the polymerase chain reaction in the correct order.

- denaturation - annealing - extension

Place the steps for the synthesis and isolation of a his-tagged protein in the correct order, with the first event at the top.

1. add sequence encoding histidine residues to the gene 2. express his-tagged protein in host cell 3. lyse host cells 4. add proteins to column containing metal resin 5. add imidazole to the column to elute his-tagged protein

Which two statements about protein purification by polyhistidine tagging are true?

A protein with six histidine residues at the N-terminus will bind to a solid matrix containing exposed nickel or cobalt atoms. A protein with six histidine residues at the C-terminus will bind to a solid matrix containing exposed nickel or cobalt atoms.

Which describes the process of electroporation?

A pulse of electricity causes small holes to form in cell membranes allowing DNA to enter

Which type of cloning vector can contain the largest amount of insert DNA?

Artificial chromosomes

Which two of the following are advantages of plasmid cloning vectors?

Autonomous replication Easy to purify

Cas9 genome editing is also referred to as

CRISPR

Genome editing techniques have been developed to exploit the CRISPR system of bacteria and archaea. Only the Blank______ component of the system is used in editing.

Cas9

To edit a eukaryotic genome by CRISPR/Cas, two components must be added to the cell, Blank______ and Blank______.

Cas9 apoenzyme guide RNA (gRNA)

Cas9 endonuclease differs from restriction endonucleases in that

Cas9 identifies a cut site by hybridizing a guide RNA

In a microbial cell, invading DNA from bacteriophage or plasmids is targeted for destruction by

Cas9 nucleaseq

Which two are characteristics of phage vectors?

Contains unique restriction sites Contains phage genes for host lysis

Oligonucleotide sequences complementary to sequences flanking the DNA sequences to be amplified in the polymerase chain reaction are called

DNA primers / primers

Which of the following are examples of synthetic biology?

Developing a bacterial strain that identifies tumor cells and releases antitumor compounds Assembling genes from plants, fungi, and bacteria in a single organism to synthesize a new biofuel

Which three properties apply to green fluorescent protein?

Encoded by a single gene From the jellyfish Aequorea victoria Easily cloned and expressed in any organism

A technique that uses an electrical field to separate DNA fragments by molecular weight through a matrix such as agarose is

Gel electrophoresis

Which technique is used to separate proteins or DNA fragments by molecular weight?

Gel electrophoresis

Which of the following statements about purifying a His-tagged protein from E. coli is false?

His-tagged proteins are easily purified by a quick centrifugation step.

Which statement about polyhistidine-tagged protein purification is true?

Most soluble cellular proteins do not bind to a solid resin with exposed cobalt or nickel atoms.

Which three statements about directed evolution are true?

Only genes of interest are targeted for mutagenesis. Altered genes may be inserted into a heterologous host. It usually involves mutating gene sequences in vitro.

When cloning, the DNA to be inserted into a vector can be obtained by Blank______ or Blank______.

PCR amplification from nucleic acid extracted and purified from an environment like soil PCR amplification from the microbe's chromosome

Which technique can amplify a specific sequence of DNA from a complex mixture of DNA sequences?

Polymerase chain reaction

Which two statements describe a selectable marker?

Provides a mechanism for discriminating between transformed and non-transformed host cells Maintains selective pressure for the presence of the cloning vector in the host cell

What is a plasmid multicloning site?

Provides multiple restriction sites for inserting DNA fragments into the cloning vector.

Restriction endonucleases (REs) differ from Cas9 endonuclease in that

REs recognize a 4-8 base pair palindromic DNA sequence

Enzymes that cut DNA at a specific sequence of nucleotides are referred to as

Restriction enzymes

Which two of the following are steps in the isolation of a his-tagged protein?

Run isolated host cell proteins through column containing metal resin. Run imidazole through the column to release his-tagged protein.

Which three of the following are components of a cosmid?

Selectable marker Multicloning site cos site

Which two characteristics of a protein favor isolation by the histidine-tagging method?

Soluble Cytoplasmic

The most commonly used thermostable polymerase in the polymerase chain reaction is ______ polymerase.

Taq

Which four of the following are characteristics of a yeast artificial chromosome?

Telomere sequences at each end Centromere sequence Multicloning site A selectable marker

Which statement regarding yeast artificial chromosomes (YACs) is false?

YACs carry an antibiotic resistance gene as a selectable marker.

In Cas9 genome editing, a guide RNA is

a component of Cas9 nuclease that specifies where to cut DNA

Following a double-stranded break by Cas9-gRNA and nonhomologous end joining repair, mutations often result because

a few bases may be lost or inserted during the repair

His-tagging is a protein purification method that involves

adding a series of histidine residues to one end of the protein

Making a large number of copies of a specific DNA sequence is referred to as

amplification

The process to produce a large number of copies of a specific DNA sequence is referred to as DNA

amplification / PCR

Which three important features are shared by most types of cloning vectors?

an origin of replication, a multi- cloning site, and a selectable marker

CRISPR loci are found in

archaea bacteria

Bacterial or yeast _______ _______ are cloning vectors that are useful for cloning very large amounts of DNA such as the amount necessary to build a genomic library.

artificial chromosomes

A DNA molecule, such as a plasmid, that carries recombinant DNA into a host cell for replication is referred to as a(n) __________ vector.

cloning

Plasmids can be introduced into bacterial cells by which three processes?

conjugation transposition electroporation

Which of the following is a hybrid cloning vector, containing DNA sequences from both plasmids and phages?

cosmid

Seamless cloning refers to

creating a recombinant DNA molecule with no evidence of restriction sites

Genetic engineering includes

crossing crop plants to improve yield breeding animals for desirable traits modifying bacteria to produce vitamins

Histidine tagging is not recommended as an approach to purify membrane-associated proteins because

cytoplasmic proteins are more successfully purified by this method

Match each step in a cycle of PCR with the appropriate temperature for the reaction to occur.

denaturation - 95 annealing - 50 synthesis - 72

Match each step in a cycle of PCR with the molecular events that occur.

denaturation - The hydrogen bonds holding the two strands of DNA are broken to produce single-stranded template DNA. annealing - Primers hydrogen bond with their complementary sequences in the template DNA. synthesis - A thermostable DNA polymerase adds dNTPs to the 3'-OH end of the primers.

The construction of production strains by targeting only genes of interest for mutagenesis is also known as _______ evolution.

directed

Two commonly used methods for introducing recombinant DNA into host organisms are ______ and ______.

electroporation chemical transformation

In each cycle of PCR, the number of target DNA molecules is doubled, meaning that the increase is

exponential

Which of the following are steps of the polymerase chain reaction?

extension denaturation annealing

A critical component of all cloning vectors is a multicloning site. Such a site is important because

foreign DNA can be placed here without interfering with replication or selection

PCR is an essential part of certain diagnostic tests in the clinical lab because

genes can be detected without having to culture the organism

The modification of the genetic code of a living organism is called

genetic engineering

A protein originally from jellyfish that can be used to study promoter activity or protein localization is the _____ _____ protein.

green fluorescent

A protein originally from jellyfish that can be used to study promoter activity or protein localization is the _____ ______ protein.

green fluorescent

CRISPRs are

groups of short palindromic repetitive sequences

Cas9 consists of a polypeptide and a

guide RNA

Plasmid copy number refers to

he number of plasmid molecules present in a cell

A gene cloned from one organism and moved into another organism, where it is transcribed and translated, is called

heterologous gene expression

One strategy for purifying an expressed protein from a mixture of bacterial proteins is to modify the protein's gene to encode a series of ___________ amino acid residues at the N- or C-terminus of the protein.

histidine

The Cas9-gRNA complex recognizes its target sequence by

hybridization with a piece of the gRNA

Sticky ends that have been created by the same restriction endonuclease can associate with each other by

hydrogen bonds

When the Cas9-gRNA complex identifies its target sequence by hybridization between the 20 base pair gRNA and the chromosome, the Cas9 nuclease

hydrolyzes phosphodiester bonds in both DNA strands

In bacterial and archaeal chromosomes, the guide RNA is encoded

in the CRISPR locus

The origin of replication allows the plasmid to replicate

independently of the host chromosome

The polymerase chain reaction (PCR) is an important tool in the clinical laboratory because

it can identify genes from samples made of complex materials like blood or soil

Taq DNA polymerase is the most commonly used DNA polymerase for PCR because

it derives from a thermophile and can withstand the temperature cycling in PCR

The guide RNA confers extreme specificity on the genome editing process because

it has a 20 base target sequence that occurs no more than once in the genome

When using PCR to obtain DNA to be cloned, it is important to remember that

knowledge of the sequence is required to create primers for PCR

In pUC19, which gene sequence is interrupted by the presence of insert DNA?

lacZ

The multicloning site in pUC19 is located in the 5' region of a gene called ______, which encodes the enzyme that cleaves lactose into glucose and galactose.

lacZ

The enzyme DNA _____________ is used to covalently join DNA fragments that have associated through complementary base pairing between their sticky ends.

ligase

To purify a His-tagged protein from an E. coli host, when the cells grow to a high density, they are

lysed with detergent or enzymes

Sequences in the CRISPR locus of a bacterial chromosome derive primarily from

mobile genetic elements

The region of a cloning vector that contains several restriction sites intended for the insertion of a DNA sequence is called the __________ site.

multicloning

Following hydrolysis of both DNA strands by a Cas9-gRNA complex, eukaryotic cells attempt to repair the damaged chromosome by

nonhomologous end joining

Plasmid copy number is determined by its

origin of replication

The ____ of ____ allows a plasmid to replicate independently of the host chromosome.

origin, replication

Engineered genomes of bacterial viruses that contain unique restriction sites and the genes necessary for host lysis are called _____ vectors.

phage

Small circular double-stranded DNA cloning vectors that replicate autonomously and are easy to purify are

plasmids

Which four of the following are major types of cloning vectors?

plasmids, viral vectors/bacteriophages, cosmids, and artificial chromosomes

The _______ _______ reaction is a technique that allows the production of a large number of copies of a specific DNA sequence from a complex mixture of sequences.

polymerase chain

Oligonucleotides that serve as the 3'-OH needed for DNA synthesis in the polymerase chain reaction are called

primers

The normal cellular function of the Cas9 nuclease is to

protect the cell from viruses

An enzyme that recognizes and cleaves specific nucleotide sequences is called a(n)

restriction endonuclease

Plasmid construction methods that use PCR instead of restriction endonucleases to specify the desired junctions between DNA molecules are termed

seamless cloning

A gene whose presence confers the ability to survive in particular conditions is called a(n)

selectable marker

Plasmids and other vectors that contain two origins of replication recognized by different host organisms are called ______ vectors.

shuttle

The field of _______ biology involves engineering biological systems to perform novel functions.

synthetic

The most commonly used thermostable polymerase in the polymerase chain reaction is _______ polymerase.

taq

To perform CRISPR/Cas9 genome editing in a eukaryotic cell, when the components are added as cloned DNA regulated by an inducible promoter,

the Cas9-gRNA complex assembles following induction

The extreme specificity that is key to Cas9 genome editing is conferred by

the guide RNA

In this image of a Cas9 nuclease, the arrow points to

the guide RNA (gRNA) (green stand)

PCR results in an exponential increase in the number of target DNA molecules, meaning that at each cycle

the number doubles

A shuttle vector contains two origins of replication. The consequence of this is that

the plasmid can replicate in two different organisms

The arrow in this image of a Cas9 nuclease points to

the polypeptide component of the Cas9 nuclease -- the outside band

Heterologous gene expression is best defined as

transfer of a gene from one organism to another, where it is expressed

Conjugation, transformation, and electroporation are all methods for

transferring a plasmid cloning vector to a microbe