E. Coli

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14 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

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Lab 5/5a

Transformation of
E. coli

with a Recombinant
Plasmid

Lab 2








Pre Lab Readiness


Familiarity and Proper use of
micropipettes


Remember the 1
st

and 2
nd

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
)



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 37
0
C

Lab 5/5a terms

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 (CaCl
2
)

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
CaCl
2
.



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+




P
-

has plasmid



no plasmid

Experimental



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

Incubate
at 37

C

If few

cells grow

If many

cells grow

colonies

lawn

Expected results

LB

LB

LB/amp

LB/amp

LB/amp/ara

P+

P
-

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