Lab 5/5a Transformation of E. coli with a Recombinant Plasmid

1. Lab 5/5a Transformation of E. coli with a Recombinant Plasmid Lab 2

2. Pre Lab Readiness • Familiarity and Proper use of micropipettes • Remember the 1st and 2nd stops • Aseptic Technique • Antibiotic Resistance • Selection markers

3. Why are we doing this? Make cells that are genetic factories for a recombinant protein Why make recombinant protein? (think insulin)

4. 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).

5. 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)

6. 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

7. 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.

8. 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. • Arabinoseis a simple sugar that is required by the bacteria to express the rfp gene.

9. 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 inwhich 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.

10. 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.

11. 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).

12. 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

13. 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

14. Labeling—Very Important! • Label two microfuge tubes P+ P- has plasmid no plasmid Experimental Control

15. 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

16. 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

17. 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

18. 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

19. If few cells grow If many cells grow Growth of E. coli Bacteria on Plates bacteria Incubate at 37C lawn colonies

20. Expected results P+ LB/amp LB/amp/ara LB P- LB LB/amp

21. 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