Microbiology Test 2 Biotechnology

_______: the use of microorganisms cells or cell components to make a product

Biotechnology

_______ ________: insertion or modification of genes to produce desired proteins

Recombinant DNA

_____: self replicating DNA used to carry the desired gene to a new cell

Vector

____: population of cells arising from one cell; each carries a new gene

Clone

1. vector (plasmid) is isolated

2. DNA containing gene of interest from a different species is cleaved by an enzyme into fragments

3. Desired gene is selected and inserted into plasmid

4. Plasmid is taken up by a cell, such as a bacterium

5. Cells with gene of interest are cloned with either of two goals in mind

(create and harvest copies of a gene) or (create and harvest protein products of a gene)

typical genetic modification procedure

_______: culture a naturally occuring microbe that produces a desired product

selection

______: mutagens cause mutations that might result in a microbe with a desirable trait

Mutation

_______ ______ ______: change a specific DNA code to change a protein (select and culture a microbe with the desired mutation)

site directed mutagenesis

______ ____: cut specific sequences of DNA; usually palindromic sequences (same read forwards as backwards)

restriction enzymes

Restriction enzymes destroy infection bacteriophage _____ in _____ cells

DNA; bacteria

Restriction enzymes CANNOT digest ____ DNA because of specific modifications in form of methylene cytosines

host

1. Restriction enzymes cut double stranded DNA at its particular recognition sites

2. These cuts produce a DNA fragment with 2 sticky ends

3. When two fragments of DNA cut by the same restriction enzymes come together, they can join by base pairing

4. The joined fragments will usually form either a linear molecule or a circular one

5. The enzyme DNA ligase is used to unite the backbones of the 2 DNA fragments producing a molecule of recombinant DNA

restriction enzyme in making recombinant DNA

Vectors carry ______ for molecular cloning

DNa

_______ _______: vectors that can exist and function in several different cell types (even prokaryotic and eukaryotic)

shuttle vectors

_____ and _____ can be used as vectors

plasmids; viruses

In ______ ____ _____ vector:

Advantages:

-uses conventional transformation/electroporation introduction

-antibiotic selection

-blue/white screening

-single strand production for sequence analysis

Disadvantages:

-Smaller DNA insert fragments than bacteriophage

typical plasmid cloning

Advantages:

-stable recombinants

-large DNA fragments

-easy to create clones and manipulate

Also includes stuffer pieces

Use of bacteriophage lambda as a cloning vector

Most common ways of inserting foreign DNA into cells

Electroporation

Transformation

______: adds DNA to cells

Transformation

______: generates electric fluid and all of pores open up to allow DNA into cells

Electroporation

Electroporation

Transformation

Virus Infection

Gene Gun

Microinjection

Protoplast fusion

Ways to get foreign DNA into cells

______ DNA: primarily bacterial. cant use for genomic DNA sequence of humans and expect it to clone because of introns in human cells

Genomic

______ DNA: eukaryotic species

Complimentary

______DNA: small tailored projects (size limited only by current cost of synthetic DNA)

Synthetic

________ _____: are made of pieces of an entire genome stored in plasmids or phages and are the most common form of bacterial libraries

Genomic libraries

Complementary DNA is made from _____ by reverse transcriptase

mRNA

complementary DNA is almost exclusively from eukaryotic organisms. Since gene expression is the desired outcome of rDNA technology, Eukaryotic genomic DNA libraries due to the presence of ____ are useless for this purpose

introns

1. A gene composed of exons and introns is transcribed to RNA by RNA polymerase

2. Processing enzymes in the nucleus remove the intron derived RNA and splices together the exon delievered RNA into mRNA

3. mRNA is isolated from the cell and reverse transcriptase is added

4. First strand of DNA is synthesized

5. The mRNA is digested by reverse transcriptase

6. DNA polymerase is added to synthesize the second strand of DNA

making complementary DNA for a eukaryote gene

Blue/White screening is a method of ______ _______ DNA.

Blue colonies: active enzyme

White colonies: no active enzyme. has foreign DNA

selecting recombinant

________ _________: uses a DNA probe to identify a cloned gene of interest

colony hybridization

______ _____:

-used becauase it is easily grown and its genomics are known

- need to eliminated endotoxins from products

-cells must be lysed to get products

E. Coli

_______ ______:

-used because it is easily grown and its genomics are known

- may express eukaryotic genes easily

Saccharomyces cerevisiae

____ cells and _____ ____:

-may express eukaryotic genes easily

-plants are easily grown

plant; whole plants

______ cells:

-may express eukaryotic genes easily

-harder to grow

mammalian

Therapeutic applications

Human enzymes and other proteins

Subunit vaccines

DNA vaccines

Gene Therapy