6 transformation

8/3/2019 6 Transformation

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Lab 5/5aTransformation of E. coli with a Recombinant

Plasmid

Lab 2



Why are we doing this?

Make cells that are geneticfactories for a recombinant protein

Why make recombinant protein?

(think insulin)



What is Genetic Transformation?

Genetic Transformation is a process in whichthe DNA of one organism is manipulated toincorporate the DNA of another organisminto its genome.

You will be transforming E.Coli bacteria witha plasmid that contains a gene for RedFluorescent 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 370C

Lab 5/5a terms



Lab 5/5a terms

• Aseptic technique is the effort taken to prevent microbialcontamination 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) toproduce a protein that disables or disrupts the effect of anantibiotic. Transforming E.coli with the plasmid pARA-R whichcarries the ampR gene (ampicillin resistant gene) renders thetransformed E.coli resistant to ampicillin.

• Selection markers are often antibiotic resistance genes whoseexpression allows for the identification of cells that have beentransformed or transfected with a plasmid or vector containing the marker gene.The ampR gene for ampicillin resistance is the selection marker in ourtransformation 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 willuse contain agar and Luria Broth.

• Agar a gelatinlike 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 thebacteria to express the rfp gene.



Lab 5/5a terms

• Calcium Chloride (CaCl2) the aqueous form of calcium chloride is usedin genetic transformation of cells by increasing the cell membranepermeability, inducing competence for DNA uptake (allowing DNA

fragments to enter the cell more readily). Calcium chloride is a salt that issolid at room temperature.

• Competent cells are bacterial cells which are capable of acceptingforeign extra chromosomal DNA. The cells we are using have been madecompetent by soaking them in CaCl2.

• Heat Shock Transformation is a basic technique in molecular biology in whichforeign plasmid DNA (pARA-R) is inserted into bacteria (E. coli ). Theprocedure consists of incubating chemically competent bacteria andplasmid DNA on ice for 10 to 15 minutes. The mixture is then placedin a 42°C water bath for 45 seconds (heat shock) then placed immediatelyback in ice.



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!

Get Ice cup and 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



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 oneminute)

42 ºC water bath



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 forminga cell wall.

-Arabinose is a simple sugar that is required by the bacteria toexpress the rfp gene



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/araplate

Use inoculating loop to spread cells



Growth of E. coli Bacteria

on Plates

bacteria

Incubate

at 37

CIf few

cells growIf 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 agriculturalproducts

Biology Cell Biology 1 d

Students know the central dogma of molecular

biology outlines the flow of information fromtranscription of RNA to translation on ribosomes

6 transformation

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