Cell and Molecular Biology Lecture 33: Exploring the Uses and Impact of Biotechnology

What is the first step in the cloning process?

Gene of interest is cut out with restriction enzyme

What is the second step in the cloning process?

Host plasmid is cut with the same restriction enzyme as gene of interest

What is the third step in the cloning process?

Gene is inserted into plasmid and ligated with ligase

What is the final step in the cloning process?

New plasmid is inserted into bacterium (transformation)

What are the two methods for bacterial transformation?

Chemical method (CaCl2) + heat shock, electroporation

What are DNA molecules that are used to transport cloned sequences between biological hosts and the test tube?

Cloning vectors?

What are the four common properties of cloning vectors?

Ability to promote autonomous replication, contain a genetic marker (usually dominant) for selection, unique restriction sites to facilitate cloning of insert DNA, minimum amount of nonessential DNA to optimize cloning

Plasmid vectors are used to clone DNA ranging in size from ______________

Several base pairs to several thousands of base pairs (100bp -10kb)

What are the disadvantages of using plasmids?

Cannot accept large fragments, standard methods are inefficient

What is phage lambda?

A bacteriophage or phage that uses E. coli as host

The lambda viral genome is a ______________ with a ____________ "sticky end" at both ends

48.5 kb linear DNA, 12 base ssDNA

Lambda viral DNA circulizes at the _________ site

cos (cohesive ends/sticky ends)

The ____________ portion of the lambda genome is not essential for replication of phage DNA and an be removed

Central

What is the size requirement for the foreign DNA when using phage lambda?

About 15,000 base pairs

What are special linear DNA vector that resemble normal yeast chromosome?

Yeast artificial chromosomes

What is the purpose of yeast artificial chromosomes?

Cloning vehicles that propogate in eukaryotic cell hosts as eukaryotic chromosomes

What size DNA can yeast artificial chromosomes clone?

Very large inserts

Yeast artificial chromosomes are __________, but their final chimeric DNA is a _________ with __________, forming an artificial chromosome

Plasmids, linear, telomeric ends

What is used to introduce new or altered genes into the genomes of human and animal cells?

Retroviral vectors

Retroviruses are ________ viruses

RNA

Viral RNA is converted into DNA by __________________

Viral reverse transcriptase

Foreign or mutated host gene introduced into the retroviral genome will be integrated into the host chromosome and can reside there for how long?

Indefinitely

What are retroviral vectors used for?

To study oncogenes and other human genes

What allows a cloned segment of DNA to be translated into protein inside a host cell?

Expression vectors

What do all expression vectors containt?

In vivo promoter and terminator, open reading frame, selection marker

Expression vectors are ______ vectors when the expression hosts are not E. coli

Shuttle

Shuttle vectors have two _______, allowing replication in two hosts

ORI (origins of replication)

What is the first step in phage display technique?

Make a combinatorial library of genes of interest

What is the second step in phage display technique?

Put the genes of interest into a vector so that each gene is expressed on the surface of a bacteriophage

What is the third step in phage display technique?

Take the collection of phages and select those with desired properties

How was CRISPR/Cas9 derived?

A natural process found in bacteria to protect themselves from pathogens

True or False: The DNA target for CRISPR/Cas9 needs to be unique and cannot appear in multiple locations

False

True or False: The single-strange guide RNA (sgRNA) can bind on either strand of DNA and Cas9 will cleave both strands, resulting in a double-strand break

True

True or False: Changing the target specificity of the RNA-protein complex does not require protein engineering but only the design of the short RNA guide

True

How could CRISPR/Cas9 be used in sickle sell anemia?

The single base mutation could be edited by CRISPR to cure the disease

How was CRISPR/Cas9 used in wheat?

Used to knock out the genes of the mildew-resistance locus