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)

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 microbial

contamination of oneself, which may result in infection,contamination of the environment you are working in, andcontamination 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 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 agrowth medium (typically agar plus nutrients) used toculture 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 primarilyused 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 used

in genetic transformation of cells by increasing the cell membranepermeability, inducing competence for DNA uptake (allowing DNAfragments 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.

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 tocreate 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 puttingthem into warm water. This is called “Heat Shock” and it creates a situation inwhich 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 aparticular 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 forcontinual 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 Successfultransformation!

• 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 plasmidExperimental 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 thebacteria’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 37CIf 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 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