Biotechnology Flashcards
Many bacteria and certain fungi contain self-replicating extrachomosomal DNA molecules called
Plasmids
Plasmid DNA is
Closed circular and double-stranded
Self-replicating and do not integrate into the host genome
Plasmids
The smallest plasmids
contain only a few thousand base pairs
The largest plasmids
contain over 100,000 base pairs
Confer a specialized and sometimes protective characteristic to the organism
Genetic information for the translation of proteins found in bacterial plasmids
Certain plasmids carry the gene for the enzyme
Beta-lactamase
Catalyzes the decomposition of certain antibiotics such as penicillin and amoxicillin
Beta-lactamase
Able to pass from an antibiotic-resistant cell to an antibiotic sensitive cell of the same or another bacterial species, thus rendering the latter resistant
Plasmids
Posess an ampicillin resistance gene and a Beta-lactamase gene
A plasmid from the strain of Escerichia coli (JM101)
One of the fastest methods to isolate plasmid DNA
Single-use columns
Based on the principle of anion exchange chromatography
Single-use columns
DNA binds to a modified
Silica matrix and is eluted using sterile distilled water
Isolation of Plasmid DNA Materials
- Pre-Lysis Buffer
- Alkaline Lysis Solution
- Neutralizing Solution
- Glassmilk Spinbuffer
- Wash Solution
- Sterile Distilled Water
- RPM Spin Filters
- Catch Tube
To resuspend the cell pellet
Add 50 micro liters Pre-Lysis Solution and mix by vortexing or pipetting up and down until completely suspended
A white precipitate will form consisting of
Cell membranes, proteins, and chromosomal DNA
To elute plasmid DNA from the Spin Filter,
Add 50 micro liters of sterile water and spin for 2 minutes to collect DNA in the bottom of the vial
Many types of bacterial can transfer ________ between individual cells.
DNA
Under certain conditions, large portions of chromosomes are transferred to the recipient cell
transferred to the recipient cell
Under some condition smaller autonomously replicating circular DNA molecules called _______ are transferred
Plasmids
Plasmid DNAs do not usually
encode information essential for the functioning of the cell
Bacteria that lack plasmids
function normally
Bacteria that possess plasmids
may be capable of performing additional functions, as for example showing resistance to specific antibodies
Some bacteria display _____________, while others do not
Competence
Ability to take up DNA at a particular stage in the life cycle
Competence
It is possible to make E. coli cells competent by treatment with
certain reagents
When the introduction of foreign DNA into a cell alters the phenotype of the cell, the cell is said to have been
Transformed
The process of introducing DNA into a cell is known as
Transformation
Most procedures for the transformation of E. coli are based on
The observation that when treated with cold CaCl2 and briefly heat-treated, bacteria could be transformed
How CaCL2 confers competence to E. Coli
Is unknown, as is the mechanism by which DNA enters the cell
A number of factors influence the efficiency of transformation including:
- The strain of bacteria
- The growth medium
- The stage of the cell cycle
In order to detect cells that have taken up plasmid DNA, it is necessary for the plasmid to contain a
Marker
A gene that when expressed as RNA or protein, reveals the presence of that gene
Marker
Frequently markers include ________ that confer resistance to antibiotics to which the cell is ordinarily sensitive
genes
Purpose of the Transformation of Plasmid lab
To demonstrate phenotype changes in bacteria that have been transformed with an antibiotic-resistance gene and a metabolic marker
E. coli cells are transformed with
The plasmid pBLU
Carries genes for two identifiable phenotypes: Amp and lacZ
Plasmid pBLU
Two identifiable phenotypes
Amp and lacZ
Responsible for bacterial resistance to Beta-lactam antibiotics
Amp gene
Codes for Beta-lactamase
Amp gene
Expresses resistance to the antibiotic ampicillin
Beta-lacatamase
Widely used reporter gene which cods for Beta-galactosidase
lacZ
5-bromo-4-chloro-3-indolyl-Beta-galactopyranoside
X-gal (lactose analog broken down by the enzyme)
Produce a visible blue product
The enzyme breaks down the lactose analog X-gal
The recipient E. coli strain lacks
amp and lacZ (thus transformed cells are converted to an amp and lacZ phenotypes
Selective agent
Ampicillin
Only _________ cells are able to grow on medium containing amipicllin
transformed
Not a selective marker, but only cells transformed with the plasmid metabolize _________ to produce blue colonies
X-gal
Luria-Bertania Media
Deionized Water Bactotryptone Bacto yeast NaCL 5M NaOH 1M HCL
