Transcript Restriction Analysis of pARA and pKAN-R

Lab 5/5a
Transformation of E. coli with a Recombinant
Plasmid
Lab 2
Pre Lab Readiness




Familiarity and Proper use of
micropipettes
– Remember the 1st and 2nd stops
Aseptic Technique
Antibiotic Resistance
Selection markers
Why are we doing this?
Make cells that are genetic
factories for a recombinant protein
Why make recombinant protein?
(think insulin)
What is Genetic Transformation?
Genetic Transformation is a process in which
the DNA of one organism is manipulated to
incorporate the DNA of another organism
into its genome.
You will be transforming E.Coli bacteria with
a plasmid that contains a gene for Red
Fluorescent Protein (RFP).
Lab 5/5a terms



What is a Plasmid?
Small circular DNA molecule
Capable of self replication
May contain an antibiotic
resistant gene(s) and/or
other gene(s)
Lab 5/5a terms
What are E. coli?
 E. coli are single cell organisms
 E. coli have a single chromosome which is a
circular DNA molecule
 E. coli live in the human intestine
 They reproduce in about 20 minutes at 370C
Lab 5/5a terms
•
Aseptic technique is the effort taken to prevent microbial
contamination of oneself, which may result in infection,
contamination of the environment you are working in, and
contamination of the samples you are working on.
•
Antibiotic Resistance the ability of a microorganism (E. coli) to
produce a protein that disables or disrupts the effect of an
antibiotic. Transforming E.coli with the plasmid pARA-R which
carries the ampR gene (ampicillin resistant gene) renders the
transformed E.coli resistant to ampicillin.
•
Selection markers are often antibiotic resistance genes whose
expression allows for the identification of cells that have been
transformed or transfected with a plasmid or vector containing the marker gene.
The ampR gene for ampicillin resistance is the selection marker in our
transformation lab.
•
Ampicillin is an antibiotic that prevents bacteria from fully forming it’s cell wall.
Lab 5/5a terms
•
Agar plates are sterile petri dishes that contains a
growth medium (typically agar plus nutrients) used to
culture microorganisms or small plants. The plates you will
use contain agar and Luria Broth.
•
Agar a gelatin like material obtained from kelp;
especially seaweed, used as a medium for growing
bacterial cultures in the laboratory.
•
Luria Broth (LB) a nutritionally rich medium primarily
used for the growth of bacteria.
•
Arabinose is a simple sugar that is required by the
bacteria to express the rfp gene.
Lab 5/5a terms
•
Calcium Chloride (CaCl2) the aqueous form of calcium chloride is used
in genetic transformation of cells by increasing the cell membrane
permeability, inducing competence for DNA uptake (allowing DNA
fragments to enter the cell more readily). Calcium chloride is a salt that is
solid at room temperature.
•
Competent cells are bacterial cells which are capable of accepting
foreign extra chromosomal DNA. The cells we are using have been made
competent by soaking them in CaCl2.
•
Heat Shock Transformation is a basic technique in molecular biology in which
foreign plasmid DNA (pARA-R) is inserted into bacteria (E. coli). The
procedure consists of incubating chemically competent bacteria and
plasmid DNA on ice for 10 to 15 minutes. The mixture is then placed
in a 42°C water bath for 45 seconds (heat shock) then placed immediately
back in ice.
How does Heat Shock allow plasmids
to enter bacteria cells?
•
Remember that plasmid DNA is negatively charged AND the plasma
membranes surrounding bacteria cells are also negatively charged. It is
relatively impossible to get the negatively charged DNA past the negatively
charged plasma membranes because like charges will repel each other.
•
When bacteria cells are made competent the positive calcium ions of the
calcium chloride solution help neutralize the negative charges of the plasmid
DNA and plasma membranes. With the negative charges neutralized, the
plasmid will have an easier time passing by the plasma membrane and getting
inside the bacteria cell.
•
To get the plasmid past the plasma membrane and inside the cell we need to
create a pressure difference between the inside and outside of the bacteria cell.
This is achieved by first getting the bacteria really cold and then quickly putting
them into warm water. This is called “Heat Shock” and it creates a situation in
which the pressure outside the cell is a tiny bit higher than the pressure inside
the cell. This pressure gradient will help to move the plasmid DNA from the
outside to the inside of the bacteria cell.
Lab 5/5a terms
•
•
•
Promoter is a region of DNA that facilitates the transcription of a
particular gene.
-Inducible promoters are promoters whose activity is induced by the
presence or absence of certain compounds, stimuli or conditions.
Inducible promoters are very powerful tools in genetic engineering
because the expression of genes linked to them can be turned on or
off by chemical or physical properties.
-Constitutive promoters are unregulated promoters that allows for
continual transcription of its associated gene.
Transcription is a chemical process that uses RNA polymerase to
convert a DNA nucleotide sequence into mRNA.
Translation is a chemical process that converts mRNA into an amino
acid sequence (protein).
Tips for a Successful
transformation!
•
Read and follow instructions!
•
Label plates properly
•
Use Aseptic Technique
•
Keep cells on ice
•
•
Time the heat shock
•
Plates go agar side up in the incubator
Overview of the Lab Procedure
Bacterial cells (E.coli) and pARA-R plasmid are mixed
E. Coli cells take up the plasmid via heat shock
Bacteria is plated on nutrient agar plates +/- amp and ara
Only cells which incorporate the plasmid DNA will grow
Labeling—Very Important!

Label two microfuge tubes
P+
has plasmid
Experimental
Pno plasmid
Control
More labeling!

Label plates on bottom (side with agar)
– LB
marked with l
– LB/amp
marked with I I
– LB/amp/ara
marked with I I I
What’s in the agar plates?
•
•
•
The LB plate contains agar and Luria Broth (LB) which is the
bacteria’s food.
The LB/amp plate contains Luria Broth (LB) and ampicillin
(amp)
The LB/amp/ara plate contains Luria Broth (LB), ampicillin
(amp) and arabinose (ara)
-Ampicillin is an antibiotic that prevents bacteria from fully forming
a cell wall.
-Arabinose is a simple sugar that is required by the bacteria to
express the rfp gene
Heat Shock

Keep P+ and P- tubes on ice for best results
–
–
–
Walk to water bath with tubes in ice bucket!
Place tubes in water bath for exactly 45 seconds
Place tubes immediately back on ice! (for at least one
minute)
42 ºC water bath
Plating and Spreading

Aliquot and plate bacteria as below
–
–
–
Spread bacteria on P- side of LB plate first then on P- side
of LB/amp plate
Discard inoculating loop
Spread bacteria on P+ side of LB plate first then on P+
side of LB/amp plate and finally over the entire LB/amp/ara
plate
Use inoculating loop to spread cells
Growth of E. coli Bacteria
on Plates
bacteria
If few
cells grow
colonies
Incubate
at 37C
If many
cells grow
lawn
Expected results
P+
LB
LB/amp
P-
LB
LB/amp
LB/amp/ara
Standards Evaluation




Biology Genetics 5 c
Students know how genetic engineering is used
to produce novel biomedical and agricultural
products
Biology Cell Biology 1 d
Students know the central dogma of molecular
biology outlines the flow of information from
transcription of RNA to translation on ribosomes