BIOL212 W5-2

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Questions and Answers

What is the primary outcome of molecular cloning?

Isolation of plasmids

Direct gene expression

Amplification of RNA

Creation of recombinant DNA (correct)

What key characteristic differentiates plasmids used as cloning vectors?

Inability to replicate independently

Uniform copy number

Restriction enzyme resistance

Copy number variation (correct)

Which enzyme is responsible for joining DNA fragments during the cloning process?

RNA ligase

Restriction endonuclease

DNA ligase (correct)

DNA polymerase

What process is NOT commonly used for amplifying DNA in cloning?

Gene expression in yeast Signup and view all the answers

What role do restriction endonucleases play in molecular cloning?

Cut DNA at specific sequences Signup and view all the answers

What is the significance of blue-white color screening in cloning?

It distinguishes between cloned and non-cloned DNA Signup and view all the answers

What is the purpose of adding a ribosome-binding site and start codon to a recombinant gene?

To initiate translation effectively Signup and view all the answers

Which of the following best describes a cloning vector?

A small, independently replicating genetic element Signup and view all the answers

Which strategy can help mitigate issues caused by recombinant proteins in host cells?

Adjusting culture and induction conditions Signup and view all the answers

What is the main benefit of using a fusion protein approach for purification?

It simplifies purification and enhances solubility Signup and view all the answers

What is the primary function of reporter genes in gene fusion studies?

To facilitate the detection of regulatory activity Signup and view all the answers

Which of the following is NOT a characteristic of protein fusions?

They involve separate transcriptional initiation Signup and view all the answers

What is the primary role of expression vectors in the cloning process?

To control the expression of cloned genes Signup and view all the answers

What component is essential for the transcription of cloned genes in T7 expression vectors?

T7 RNA polymerase Signup and view all the answers

Which method is commonly used to optimize gene expression in a bacterial host?

Codon optimization Signup and view all the answers

Why must ribosome-binding sites be engineered into vectors for eukaryotic gene expression?

Eukaryotic mRNAs cannot bind to prokaryotic ribosomes Signup and view all the answers

What is the role of Isopropyl β-D-1-thiogalactopyranoside (IPTG) in the expression of cloned genes?

To induce expression of the lac operon Signup and view all the answers

When expressing eukaryotic genes in prokaryotic systems, what modification is commonly made to facilitate the process?

Use of cDNA derived from mRNA Signup and view all the answers

What defines a strong promoter in the context of expression vectors?

A high affinity for RNA polymerase Signup and view all the answers

What is a common characteristic of plasmids utilized in gene cloning?

They replicate independently of chromosomal DNA Signup and view all the answers

Which strain of E. coli is specifically designed to work with T7 expression vectors?

BL21(DE3) Signup and view all the answers

What benefit do weakly pathogenic recombinant strains of Listeria monocytogenes provide in cancer therapy?

They are cleared by healthy cells but not by tumor cells. Signup and view all the answers

Which of the following describes the process of environmental gene mining?

Directly isolating and cloning genes from environmental DNA. Signup and view all the answers

What is a key characteristic of the enzymes isolated through gene mining for industrial applications?

They can withstand extreme conditions such as high temperatures. Signup and view all the answers

How does the engineered protective antigen from Bacillus anthracis function in anticancer therapy?

It delivers synthetic antibodies to intracellular cancer targets. Signup and view all the answers

Which application of hyperstable enzymes has been highlighted in the context of food industry cleaning processes?

They perform as well or better than conventional cleaning methods. Signup and view all the answers

What is a major benefit of using genetically engineered microorganisms for producing mammalian proteins?

They significantly decrease the costs of protein purification. Signup and view all the answers

Which feature of the Ti plasmid is crucial for transferring genes into plant cells?

The T-DNA segment at the plasmid's ends. Signup and view all the answers

What characteristic distinguishes a transgenic organism?

It has at least one gene from a different species integrated into its genome. Signup and view all the answers

Which statement correctly describes human somatotropin?

It is synthesized from cloned cDNA transcribed from mRNA. Signup and view all the answers

What is the primary role of recombinant bovine somatotropin (rBST) in agriculture?

To increase milk production in dairy cows. Signup and view all the answers

Which of the following is a component of the binary vector system used for gene transfer to plants?

Cloning vector with multiple cloning sites. Signup and view all the answers

What distinguishes the use of the Ti plasmid in genetic engineering compared to other plasmids?

It is derived from a plant pathogen and facilitates gene transfer. Signup and view all the answers

Which feature distinguishes A.tumefaciens in the context of plant genetic engineering?

It facilitates the transfer of foreign DNA into broadleaf plants. Signup and view all the answers

What is one primary limitation of the Ti system when used for genetic engineering?

It cannot be used for monocot transformation. Signup and view all the answers

What role does the Bt toxin play in genetically modified plants?

It acts as an insecticide against specific larvae. Signup and view all the answers

Which type of vaccine uses a harmless carrier virus to induce an immune response?

Vector vaccine Signup and view all the answers

Which of the following represents a common target for genetic modification in crops?

Herbicide and insect resistance Signup and view all the answers

What type of plant is primarily improved using alternative transformation methods like particle bombardment?

Cereal crops Signup and view all the answers

What is a defining characteristic of subunit vaccines?

They include only specific proteins from a pathogen. Signup and view all the answers

What is a potential controversy surrounding genetically modified crops like those engineered for glyphosate resistance?

Their potential carcinogenic effects. Signup and view all the answers

Which of the following best describes a polyvalent vaccine?

A vaccine that protects against multiple diseases. Signup and view all the answers

What is the mechanism by which glyphosate inhibits weed growth in crops like soybeans?

It blocks the biosynthesis of aromatic amino acids. Signup and view all the answers

What is a notable feature of the Plasmodium infection cycle related to its vector?

It involves Anopheles mosquitoes as transmission vectors. Signup and view all the answers

Which of the following symptoms is NOT associated with a Plasmodium infection?

Severe itching Signup and view all the answers

What is a critical method used for diagnosing a Plasmodium infection?

Identifying Plasmodium-infected erythrocytes in blood smears Signup and view all the answers

Which preventive measure is key in controlling malaria transmission?

Using treated bed nets to prevent mosquito bites Signup and view all the answers

What is the primary role of the Semi-Synthetic Artemisinin Project?

To synthesize artemisinic acid via engineered microorganisms Signup and view all the answers

Which type of biofuel is produced through the fermentation of corn sugar by yeast?

Ethanol Signup and view all the answers

What is the main advantage of thermophiles in biofuel production?

Higher temperature tolerance Signup and view all the answers

Which engineered organism converts glucose into propane?

Escherichia coli Signup and view all the answers

What bioactive compound do microalgae such as Chlorella primarily produce?

Triacylglycerides Signup and view all the answers

What process is required for breaking down switchgrass into fermentable sugars?

Pretreatment Signup and view all the answers

Which of the following is NOT a current major biofuel?

Gasoline Signup and view all the answers

Which enzymes are utilized from Bacillus subtilis to increase octane in hydrocarbon production?

Fatty acid elongation enzymes Signup and view all the answers

Which statement accurately describes the primary benefit of synthetic biology in product development?

It produces novel products with minimal environmental impact compared to chemical methods. Signup and view all the answers

What is the significance of using engineered E.coli and yeast for vanillin synthesis?

They can produce vanillin more cheaply than chemical methods. Signup and view all the answers

Which of the following correctly identifies a challenge associated with the economic synthesis of artemisinin?

The natural production process is too costly and time-intensive. Signup and view all the answers

What is a key motivation behind the International Genetically Engineered Machine (iGEM) competition?

To foster new innovations in synthetic biology among students. Signup and view all the answers

Which pathway correctly describes the conversion process for vanillin biosynthesis in engineered E.coli?

Tyrosine → Ferulic acid → Vanillin Signup and view all the answers

What is one of the main reasons artemisinin cannot be economically synthesized chemically?

It possesses an intricate structure that poses synthetic challenges. Signup and view all the answers

Which characterization is most accurate for the disease malaria caused by Plasmodium spp.?

It is a protist disease with a significant global health impact. Signup and view all the answers

What is a potential environmental concern related to the production and use of synthetic vanillin?

It could threaten traditional farming practices in vanilla-producing regions. Signup and view all the answers

What feature of artemisinin’s natural source makes it significant in traditional medicine?

Its antimalarial properties have been recognized for centuries. Signup and view all the answers

Why is the use of genetic engineering considered a critical advancement in synthetic pharmaceuticals?

It can create sustainable methods for producing complex natural products. Signup and view all the answers

What is the role of the protospacer adjacent motif (PAM) in the genome editing process?

It allows Cas9 to locate and bind to the target DNA sequence. Signup and view all the answers

In CRISPR technology, what is a possible consequence of the DNA break repair process?

It can induce both insertions and deletions, causing gene mutations. Signup and view all the answers

What ethical concerns are associated with CRISPR technology as demonstrated in the case of the CRISPR babies?

The unknown risks and potential negative consequences of genome editing. Signup and view all the answers

How does homologous recombination assist in CRISPR genome editing?

It incorporates new DNA using a template sequence, allowing for precise edits. Signup and view all the answers

What was the unexpected result from the mutation of the SlAGO7 gene in tomato plants?

It caused the mutation to produce needle-like or wiry leaves. Signup and view all the answers

What is the primary role of the CRISPR system in prokaryotic organisms?

Maintaining genome stability and evading viral destruction Signup and view all the answers

Which component is essential for the transcribing process in the CRISPR interference mechanism?

Cas proteins Signup and view all the answers

Why are spacer sequences in the CRISPR system biologically significant?

They act as a memory bank for previous viral invasions. Signup and view all the answers

When did Francisco Mojica first characterize what is now known as a CRISPR locus?

1993 Signup and view all the answers

What distinguishes the CRISPR RNA (crRNA) in the interference mechanism?

It base-pairs with foreign nucleic acids to lead to their destruction. Signup and view all the answers

What was significant about the palindromic repeats discovered by Yoshizumi Ishino et al. in 1987?

They provided insight into a prokaryotic adaptive immune system. Signup and view all the answers

Which of the following proteins is NOT a component associated with CRISPR systems?

tRNA Signup and view all the answers

What is the primary function of the crRNA in the CRISPR system?

To provide a pattern recognition sequence Signup and view all the answers

Which statement accurately describes the distribution of CRISPR in organisms?

Found in approximately 90% of Archaea and 70% of Bacteria Signup and view all the answers

What role does Cas9 play in the CRISPR mechanism?

It acts as an endonuclease to cleave nucleic acids Signup and view all the answers

Which event can allow bacteriophages to evade recognition by CRISPR systems?

Mutating their genomes Signup and view all the answers

What is a significant innovation reported by Charpentier and Doudna in 2012 related to CRISPR?

Fusing crRNA and tracrRNA into a single synthetic guide RNA Signup and view all the answers

How does the CRISPR system achieve the induction of double-stranded breaks in DNA?

Through recognition of specific DNA sequences by Cas proteins Signup and view all the answers

What technique is primarily used to process the RNAs involved in the CRISPR mechanism?

RNase III cleavage Signup and view all the answers

Which group first reported the application of CRISPR genome editing in human cells?

Feng Zhang and George Church's groups Signup and view all the answers

What is the primary purpose of the PAM sequence in the CRISPR system?

To facilitate the binding of Cas proteins to target DNA Signup and view all the answers

Study Notes

Tools of the Genetic Engineer

Genetic engineering uses various tools for manipulating DNA.
These tools include PCR (Polymerase Chain Reaction) and nucleic acid hybridization.
Molecular cloning moves a gene from its original source to a smaller, more manageable genetic element (vector).
This creates recombinant DNA.

Molecular Cloning

Molecular cloning involves moving a gene from its original source to a small, manageable genetic element called a vector.
The result is recombinant DNA.
Gene manipulation and replication of cloned DNA are possible outcomes.
Cloning can be used to produce proteins or DNA for research purposes and various industrial applications.

Expressing Foreign Genes in Bacteria

Expression vectors are used to control the expression of cloned genes.
Regulation of gene expression is typically done at the transcriptional level.
Strong promoters, like those from bacteriophages (e.g., T7 promoter), with T7 RNA polymerase are often used.
Expression vectors place cloned genes under the control of specific promoters, like the T7 promoter.
An example of a useful system is using E. coli (bacteria) strains (like BL21 (DE3)) that are engineered for use with pET T7 expression vectors and induced using Isopropyl β-D-1-thiogalactopyranoside (IPTG), a molecular mimic of allolactose in the lac operon of E. coli.

Molecular Methods for Mutagenesis

Mutagenesis is the process of creating mutations in organisms or DNA.
Molecular methods for this process are tools utilized in genetic engineering to achieve that.

Reporter Genes and Gene Fusions

Reporter genes' coding sequences are fused with regulatory regions from other sources for studying gene regulation.
Researchers can monitor the activity of the regulatory region by testing the reporter's activity under different conditions.
An example is using a reporter gene's output to identify when a particular regulatory region is activated or deactivated.

Cloning Vectors

Cloning vectors come in various types and have varying copy numbers.
Selection of the proper vector depends on the size of the DNA fragment that needs to be cloned.
Plasmids, like pUC19, are common cloning vectors.

Plasmids

Plasmids are small, self-replicating DNA molecules often used as cloning vectors.
They have variable copy numbers.
Plasmid compatibility refers to the ability of different plasmids to replicate in the same cell without causing problems.

Hosts for Cloning Vectors

Bacteria like Escherichia coli and Bacillus subtilis and yeast (eukaryotes) like Saccharomyces cerevisiae are commonly used as hosts for cloning vectors.
E. coli are well-characterized, and easy to grow, but genetically less stable than eukaryotes.
Bacillus subtilis easily transformed.
Eukaryotic hosts, such as yeast, are good for expressing eukaryotic genes but require different methods from bacterial hosts.

How Genes are Cloned

DNA can be amplified or synthesized to create fragments.
DNA fragments are inserted into cloning vectors, often by using restriction enzymes.
DNA ligase joins or anneals the inserted DNA into the vector.
The cloned DNA is then introduced into a host organism.
Host cells are selected using specific properties, such as antibiotic resistance or blue/white screening (based on the lac operon).

Codon Optimization

Adjusting mRNA to optimize translation in the host organism is done through codon optimization.
Modification of codon usage in cloned genes is done to match the codon usage of the host organism to improve expression.

Protein Stability and Purification

Recombinant proteins can cause issues for host cells like degradation, toxicity, or insoluble inclusion.
Adjusting culture conditions for the host, such as the temperature used for protein expression (e.g., 18°C), altering the host to make the recombinant protein more stable, or using fusion proteins can improve protein stability and purification.
Fusion of protein genes linked with a protease cleavage site will combine two genes into one purified protein.
Methods like using a carrier protein can be used for protein purification to simplify or reduce challenges in the purification process.