Lab Day 1 Pre-Reading Flashcards
what is a vortex used for
it is for mixing small volumes
what is a microcentrifuge used for
it is used for rapid separation of small quantities of cells and large molecules and/or precipitates
what is the purpose of recombinant DNA technology
it is a set of tools that allows molecular biologists to create novel DNA molecules that do not exist in nature, and to produce large quantities of “recombinant DNA” molecules
what is recombinant DNA
a DNA strand that is artificially created by combining DNA from two or more sources.
what was the first important contribution of recombinant DNA technology to medicine from Genetech in 1978
the company cloned the human insulin gene into a plasmid vector that allowed the human insulin protein to be “grown” or propagated within E. Coli
what was the first recombinant DNA product apptoved by the FDA
recombinant human insulin, or humulin
what is a DNA vector
it is a vector that acts as a vehicle for a specific DNA segment that inserted into the vector
what is the recombinant plasmid vector and what do we do with it
the recombinant plasmid vector carries the foreign DNA segment we are interested in, we will introduce it to a living organism where it will be replicated and studied
what foreign DNA are we interested in
we are interested in a portion of the 16s ribosomal RNA gene
what is the overall goal of the river project
determine the diversity of the microbes influencing the water quality
why do we want to know the diversity of the microbes in the river
microbes are a source of contaminants but are beneficial by detoxifying pollutants.
by identifying the species of microbes, we will be able to evaluate the health of the watershed system
what is our role in the river project
we will clone the 16rRNA gene fragment that was previously PCR amplified
What does clone mean
to clone something means that we will insert the PCR fragment into a vector and recover the recombinant plasmid from the bacterial cells that are identical
how are the bacterial cells identical
they have identical DNA as they were derived from a single ancestral cell
why do we work with e coli (4 reasons)
they are easy to culture and are not harmful to humans
they are versatile and grow at a range of temperatures (optimal at 37 C)
they utilize sugars as carbon and energy sources
they can manufacture most of the organic molecules they require when provided with sugar, water, and salts
what are the characteristics of Ecoli
they are rod shapes, they are 1/500th the volume of a typical eukaryotic cell
what is important about the cell wall in e-coli
they are made of carbohydrates and proteins
e coli is gram-negative, meaning it has less peptidoglycan content and cannot be stained
why is E coli suitable for genetic studies (5 reasons)
it is easily grown in asexual cycles in 20 minutes
generally has 1 gene per trait
can have many different colonies on a Petri plate
can participate in gene exchange by conjugation, transformation, and transduction
serves as a host for bacteriophages and plasmids
what is a plasmid good for
it is an excellent vector for carrying foreign genes
what is a plasmid
it is a small double-stranded circle of DNA
why are plasmids good vectors
they are self replicating and bacteria will spontaneously take up plasmids through transformation
what type of information do plasmids carry
they carry extra information not normally needed for cell survival, such as antibiotic resistance and toxic proteins used for host invasions
describe the steps for producing recombinant plasmids
- the bacterial plasmid is manipulated with enzymes
- using a restriction enzyme, the double-stranded DNA of the plasmid will be broken and the gene of interest (16sRNA) will be introduced into the tube along with ligase to seal the DNA molecules together
this makes a recombinant plasmid molecule
- the recombinant plasmid is introduced to E-coli by transformation
- The transformed E-coli cells will replicate along with the plasmid and can produce recombinant protein
- The cells are lysed open and the plasmid DNA or protein of interest is purified
how is e coli coaxed into taking up the plasmid vector
we add calcium chloride and the temperature is elevated by heat shock
how long does it take to double the amount of recombinant DNA or protein in an e-coli cell
20 minutes
what are the three features that all commerically produced plasmids share
1.) origin of replication, 2) selectable marker, 3.) cloning site
what is the purpose of the origin of replication
site where the DNA replication machinery recognizes
plasmid is autonomous and replicates to high copy number within the cell (1,000 per cell)
what is the selectable marker
since the efficiency of transformation is low, we use selectable markers that only allows cells that picked up the plasmid to be selected
give an example of a selectable marker
antibiotic-resistant genes
for example, many plasmids have kanamycin resistance genes.
After transformation, cells are grown on a media with the kanamycin antibiotic.
The cells that did NOT pick up the plasmid will die and not grow
The cells that picked up the plasmid are SELECTED and will grow as they have the resistant gene
what is a cloning site
restriction enzymes only cut at a specific site, the cloning site or cloning cassette provide multiple sites for restriction enzymes to cut, giving the researcher flexibility
what is a sticky end
sticky ends are 5’ or 3’ overhangs that allow the DNA to anneal (H-bond) even though it is not covalently bonded yet
