Lab 5/5a

Transformation of E. coli with a Recombinant Plasmid

Lab 2

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 its 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 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 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 42C water bath for 45 seconds (heat shock) then placed immediately back 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

LabelingVery Important!

Get Ice cup and 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

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 C water bath

Whats in the agar plates?

The LB plate contains agar and Luria Broth (LB) which is the bacterias 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

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

Incubate at 37C

If many cells grow

colonies

lawn

Expected results
P+

LB

LB/amp

LB/amp/ara

P-

LB

LB/amp

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