WEEK 9 ASSIGNMENT MCB3020 BACUSMO

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 his residues to the gene 2, Express his-tagging protein in host cell 3. lyse host cell 4. add proteins to column containing metal resin 5. add imidazole to the column to elute his-tagging protein

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

1. Denaturation 2. Anealing 3. Extension

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

A protein with six histidine residues at the C-terminus will bind to a solid matrix containing exposed nickel or cobalt atoms. A protein with six histidine residues at the N-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 four of the following are characteristics of a yeast artificial chromosome?

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

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

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

Bacteriophages Plasmids Cosmids Artificial chromosomes

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

Blank 1: Primers or DNA primers

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.

Blank 1: artificial Blank 2: chromosomes or chromosome

Fill in the blank question. The construction of production strains by targeting only genes of interest for mutagenesis is also known as ________ evolution.

Blank 1: directed

Fill in the blank question. A protein originally from jellyfish that can be used to study promoter activity or protein localization is the _________ __________ protein.

Blank 1: green Blank 2: fluorescent, fluorescence, flourescent, or flourescence

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.

Blank 1: histidine or his

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

Blank 1: polymerase or DNA Blank 2: chain or amplification

Cas9 genome editing is also referred to as Blank______.

CRISPR

Cas9 endonuclease differs from restriction endonucleases in that Blank______.

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 Blank______.

Cas9 nuclease

Which two are characteristics of phage vectors?

Contains unique restriction sites Contains phage genes for host lysis

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

Cosmid

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

Cytoplasmic Soluble

Which enzyme forms phosphodiester bonds between cloned DNA fragments and the cloning vector?

DNA ligase

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

Denaturation: 95°C Annealing: 50°C Synthesis: 72°C

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?

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

Which two statements describe a selectable marker?

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

Which statement about polyhistidine-tagged protein purification is true? A protein with six consecutive histidine residues preferentially binds to a solid resin with exposed iron or nickel atoms. Most soluble cellular proteins do not bind to a solid resin with exposed cobalt or nickel atoms. A protein with six histidine residues anywhere in its sequence will bind to a solid resin with exposed cobalt or nickel atoms

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

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

Multicloning site Origin of replication Selectable marke

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

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

Polymerase chain reaction

What is a plasmid multicloning site?

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

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

RESTRICTION ENDONUCLEASES

Restriction endonucleases (REs) differ from Cas9 endonuclease in that Blank______.

REs recognize a 4-8 base pair palindromic DNA sequence

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 statement regarding yeast artificial chromosomes (YACs) is false? YACs have a telomere at each end. YACs carry an antibiotic resistance gene as a selectable marker. YACs replicate once per cell cycle. YACs have a multicloning site where foreign DNA may be inserted.

YACs carry an antibiotic resistance gene as a selectable marker

In Cas9 genome editing, a guide RNA is Blank______.

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 Blank______.

a few bases may be lost or inserted during the repair

His-tagging is a protein purification method that involves Blank______.

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

CRISPR loci are found in Blank______ and Blank______.

bacteria archaea

Genetic engineering includes Blank______. [Choose three.]

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

Fill in the Blank Question Fill in the blank question. 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 electroporation transformation

Seamless cloning refers to Blank______.

creating a recombinant DNA molecule with no evidence of restriction sites

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

cytoplasmic proteins are more successfully purified by this method

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

doubles

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

exponential

A critical component of all cloning vectors is a multicloning site. Such a site is important because Blank______. Multiple choice question.

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

Fill in the blank question. A technique that uses an electrical field to separate DNA fragments by molecular weight through a matrix such as agarose is

gel electrophoresis

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

genes can be detected without having to culture the organism

CRISPRs are Blank______.

groups of short palindromic repetitive sequences

Cas9 consists of a polypeptide and a Blank______.

guide RNA

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

guide RNA (gRNA) Cas9 apoenzyme

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

heterlogous something

The Cas9-gRNA complex recognizes its target sequence by Blank______.

hybridization with a piece of the gRNA

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

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 Blank______.

hydrolyzes phosphodiester bonds in both DNA strands

In bacterial and archaeal chromosomes, the guide RNA is encoded Blank______.

in the CRISPR locus

The origin of replication allows the plasmid to replicate Blank______.

independently of the host chromosome

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

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 Blank______.

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 Blank______.

knowledge of the sequence is required to create primers for PCR

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, lacZ-alpha, or lacZa

Once the sticky ends of the cloned DNA and cloning vector have formed complementary base pairs by hydrogen bonding, the two fragments can be covalently joined by adding the enzyme DNA _________ to the reaction mixture.

ligase

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 Blank______.

lysed with detergent or enzymes

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

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 ________

multicloning, multi-cloning, polylinker, or MCS

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

nonhomologous end joining

Plasmid copy number is determined by its Blank______.

origin of replication

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

phage

Fill in the Blank Question Fill in the blank question. Small circular double-stranded DNA cloning vectors that replicate autonomously and are easy to purify are _____

plasmids

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

primers

The normal cellular function of the Cas9 nuclease is to Blank______.

protect the cell from viruses

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

seamless cloning

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

selectable marker

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

shuttle

Fill in the blank question. 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

taq polymerase

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

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

the Cas9-gRNA complex assembles following induction

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

the guide RNA

A shuttle vector contains two origins of replication. The consequence of this is that Blank______. Multiple choice question.

the plasmid can replicate in two different organisms

Heterologous gene expression is best defined as Blank______.

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

Conjugation, transformation, and electroporation are all methods for Blank______.

transferring a plasmid cloning vector to a microbe

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

Which of the following statements about purifying a His-tagged protein from E. coli is false? His-tagged proteins are generally expressed from a plasmid vector. His-tagged proteins are easily purified by a quick centrifugation step. The E. coli host cells are lysed by treatment with detergent or enzymes. His-tagged proteins are mixed with all other soluble E. coli proteins.

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

Which three of the following are components of a cosmid?

Multicloning site Selectable marker cos site

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

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

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

electroporation chemical transformation