Cell Biology Lecture 33: Uses and Power of Biotechnology

(Cloning Process) What is the first step of the cloning process?

Gene of interest is cut out with restrictive enzyme

(Cloning Process) What is the second step of the cloning process?

Host plasmid is cut with the same restrictive enzyme that cut the gene of interest

(Cloning Process) What is the third step of the cloning process?

Ligation of DNA sample and plasmic DNA

(Cloning Process) What is the fourth step of the cloning process?

Transformation

(Cloning Process) What is the fifth step of the cloning process?

Growth on agar plates

(Cloning Process) What is the process of transferring exogenous DNA into cells called?

Transformation

(Cloning Process) What are the two general methods for transforming bacteria?

1. CaCl2 + Heat Shock

2. Electroporation

(Cloning Process) What do blue colonies on the agar plate represent?

Bacteria that contain pVector

(Cloning Process) What do blue colonies on the agar plate express?

Functional alpha fragment from intact LacZ

(Cloning Process) What do white colonies on the agar plate represent?

Bacteria that contain pInsert

(Cloning Process) Do white colonies produce LacZ alpha fragments?

No

(Cloning Vectors) What are cloning vectors?

DNA molecules used to transport cloned sequences from host to test tube

(Cloning Vectors) What are the four common properties of cloning vectors?

1. Ability to promote autonomous replication

2. Genetic marker

3. Unique restriction sites

4. Minimum nonessential DNA

(Cloning Vectors) What are plasmid vectors used for?

Cloning DNA with ranging size (100bp - 10kb)

(Cloning Vectors) What are the disadvantages of using plasmid vectors?

1. Cannot accept large fragments

2. Inefficient methods of transformation

(Cloning Vectors) What is a phage that uses E. coli as a host?

Phage Lambda

(Cloning Vectors) What is the genome structure of Phage Lambda?

48.5 kb linear DNA with 12 base ssDNA sticky ends

(Cloning Vectors) What are the properties of the sticky ends of phage lambda?

1. Complementary to each other

2. Can hybridize to each other

(Cloning Vectors) What are the sticky ends of phage lambda called?

Cos site

(Cloning Vectors) Where does DNA circularize on the phage lambda?

Cos site

(Cloning Vectors) Do phage lambda or plasmids allow for larger genomes?

Phage lambda

(Cloning Vectors) What do yeast artificial chromosomes act as?

Propagate as chromosomes

(Cloning Vectors) What are yeast artificial chromosomes able to clone?

Very large inserts of DNA (100 kb - 10 Mb)

(Cloning Vectors) What are yeast artificial chromosomes classified as?

Plasmids

(Cloning Vectors) What is the final chimeric DNA for yeast artificial chromosomes?

Artificial chromosome

(Cloning Vectors) What is an artificial chromosome?

Linear DNA molecule with telomeric ends

(Cloning Vectors) What are retroviral vectors used for?

Introducing new/altered genes into genomes

(Cloning Vectors) What are retroviruses classified as?

RNA viruses

(Cloning Vectors) What converts viral RNA into DNA?

Viral reverse transcriptase

(Cloning Vectors) What happens after viral RNA is converted to DNA?

Integrated into host genome

(Cloning Vectors) Can foreign/mutated genes be introduced into the host chromosome through retroviral vectors?

Yes

(Cloning Vectors) How long can foreign/mutated genes introduced into the host chromosome through retroviral vectors remain?

Indefinitely

(Cloning Vectors) What are retroviral vectors used to study?

Oncogenes

(Cloning Vectors) What do expression vectors allow?

Cloned segment of DNA be translated into protein

(Cloning Vectors) What do expression vectors contain?

1. in vivo promotor and terminator

2. Open reading frame (ORF)

3. Selection marker

(Cloning Vectors) What are expression vectors when the host is not E. coli?

Shuttle vectors

(Cloning Vectors) What do shuttle vectors have that allows for replication in two hosts?

Two origins of replication

(Cloning Vectors) What are the bacterial expression vectors?

Plasmids and phages

(Cloning Vectors) What are the yeast expression vectors?

1. Plasmids

2. Yeast Artificial Chromosomes

(Cloning Vectors) What are the mammalian expression vectors?

1. Retroviral Vectors (Best)

2. Plasmid

(Phage Display) What does phage display make?

Combinatorial library of genes of interest

(Phage Display) Where are gene products expressed in phage display?

Surface of bacteriophage

(Phage Display) Where are genes placed in phage displace?

Into a vector (inside phage)

(Phage Display) What are selected from the gene products expressed in phage display?

Those with desired properties

(Genomic Editing) What is CRISPR derived from?

Natural process in bacteria to protect themselves from pathogens

(Genomic Editing) What does CRISPR target?

Genes for editing and regulating

(Genomic Editing) Does the DNA target need to be unique for CRISPR?

No

(Genomic Editing) Can the DNA target appear in multiple locations for CRISPR?

Yes

(Genomic Editing) What targets the DNA target for cleavage in CRISPR?

Cas9 Nuclease

(Genomic Editing) What is the result of Cas9 cleaving both sides of DNA in CRISPR?

Double Strand Break (DSB)

(Genomic Editing) What does double strand break result in for CRISPR?

Silencing of DNA sequence

(Genomic Editing) Does CRISPR require protein engineering?

No

(Genomic Editing) Is protein engineering needed to change the target specificity of the RNA-protein complex?

No

(Genomic Editing) What is the role of single guide RNA (sgRNA)?

To find and bind to DNA sequence

(Genomic Editing) What is needed to change the target specificity of the RNA-protein complex?

Design of the short RNA guide

(Genomic Editing) What human disease is treated with CRISPR?

Sickle Cell Anemia

(Genomic Editing) What is CRISPR used in farming?

Removing genes of mildew-resistance locus in wheat

(Genomic Editing) What is CRISPR used in genome changing?

Germline manipulation