Biotechnology: Bacterial Transformation Lab

How can we manipulate inheritable information?

You are going to be split into one of the following groups

• DNA cloning and applications• Different methods used to study DNA• Medical applications of DNA technology• Agricultural applications (plants & animals)• Safety and Ethical issues surrounding the

applications of DNA technology

You will create a poster about your topic & present it to the class.

Genetic information passed from parent to offspring via DNA provides for

continuity of life.

• In order for DNA to direct cellular activities it must first be transcribed.

• Some of the RNA’s are used immediately for ribosomes or to control other cellular processes.

• Other RNA’s are translated into proteins that have important roles in determining metabolism & development.

• When the DNA of a cell changes, the RNA’s and proteins they produce often change, which in turn changes how the cell functions.

• DNA can change in different ways:– Mutated (spontaneously, environmental effects or

DNA replication error)– Biotechnologists can cause an intentional change

Biotechnologists have a most powerful tool:

• They have the ability to transfer the DNA of one organism into another & make it function in the new organism.

• With this ability they can make cells produce novel protein products that the cells did not make previously.

Fredrick Griffith Experiment

CONCLUSION: The living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells that allowed the R cells to make capsules.

What they do today…

• Biotechnologists have succeeded in inserting a gene (Bt) from the bacterium:– Bacillus thuringiensis

into the corn genome.• When expressed the Bt gene produces a toxin

that kills caterpillars & controls earworms that damage corn

Applications for genetic engineering

Human manipulation of DNA has raised several ethical, social, and medical issues

Working with bacteria

What’s a plasmid?A plasmid is a small circular piece of double-stranded DNA that has an origin of replication.

Natural Transformation

Cells have surface proteins that bind to DNA and bring it into the cell. Then the orig. DNA and the new DNA are compared. If there is enough similarities the new DNA will be welcomed. If there isn’t then the new DNA is digested by the cellular enzymes.

Why is this beneficial to bacteria?

Gives them more genetic material which may increase their chances of surviving in a changing environment.

Why is transformation useful to research scientists?

• Allows for further study or work with genes on the plasmid DNA and the proteins that the genes code for.

• Enables researchers to manipulated and study genes already present in the bacterial genome.

Should you plate some of your transformed bacteria onto plates with

antibiotics? Why or why not?

• Yes, this ensures that those ;bacteria that take up the plasmid will retain it and allows you to select for those bacteria that have actually taken up the plasmid.

What would you expect to see if you plated some of your

transformed bacteria onto a plate without antibiotic? Would there be an advantage to doing this (in terms of understanding

your results)? Explain

• You would see a “lawn”. Yes, this would allow you to assess cell viability. How viable the cells are is important to know in the event that there are no colonies on the plate that does contain antibiotic.

• In addition, being able to directly contrast the lawn on the antibiotic-free plate with the colonies present on the plate with antibiotic is a good demo. Of how small a % of cells actually took up the plasmid.

• Contrast also indicates that the antibiotic is good.

• To transform bacteria with plasmids, technicians first make the bacteria competent (capable of taking up DNA) by placing them in calcium chloride and chilling them.

• Plasmid is then added to the competent bacteria and the plasmid/bacteria combo. Is taken through a few more steps to make the bacteria take up the DNA.

• In your experiment, should you treat a tube of bacteria that your don’t add plasmid to exactly as your do the tube of bacteria that you will transform?

• Why or why not?

• Treating bacteria that you have not added plasmid to exactly as you do the bacteria that you add plasmid to, provides a control demonstrating that the antibiotic-resistant colonies that appear on the plate are a result of the plasmid being taken up by the cells.

• In the absence of plasmid, there should be no antibiotic-resistant colonies present on the plate.

Let’s work with bacteria!!!!

LAWN

COLONIES

TOMORROW

Rendering bacteria competent

• Changing the ionic strength of the medium and heating the cells in the presence of positive ions (usually calcium).

• This treatment renders the cell membrane permeable to DNA.

• Recently high voltage has also been used to render cells permeable to DNA– electroporation

• Once the DNA has been taken in by the cell, the use of that DNA by the cell is referred to as expression

• The expression of the new DNA depends on its integration with the host DNA.

• Normally scientists want to introduce DNA that is not similar to the host DNA & therefore the cell would destroy the introduced DNA.

• Scientists have found a way around this by introducing the new DNA as plasmid DNA.– Does not have to be similar to the host DNA

Selecting for transformed bacteria

• 2 problems to overcome:– Cells containing plasmids reproduce at a slower

rate & the pressure is great to rid themselves of the plasmid.• To overcome this there needs to be an advantage

– We have to be able to determine which bacteria have received the plasmid.

Solutions to the problems

• Scientists use a system involving antibiotics and genes for resistance of antibiotics.

• In a typical transformation, billions of bacteria are treated and exposed to plasmid DNA.

• Only a fraction (less than 1 in 1000) will acquire the plasmid.

• Antibiotic resistance genes provide a means of finding the bacteria that took in the plasmid.

• The treated bacteria are then exposed to the antibiotic.

• The bacteria that took in the plasmid will grow on the antibiotic plate.

• Resistance to an antibiotic is known as a selectable marker; we can select for cells that contain it.

• Other marker genes are color marker genes, which change the color of a bacterial colony.

Green Fluorescent Protein (GFP)

This is an example of a color marker scientists use.

Color markers used as reporter molecules

• If you wanted to know what a certain protein did or where it went you could attach the color marker.

• Let’s say you wanted to know if a particular protein had anything to do with making blood vessels. If the blood vessels started glowing you would have your answer.

Suppose you have a plasmid that contains both the gene for GFP and a gene for resistance to ampicillin

(an antibiotic), how will you be able to determine if the bacterial

cells have been transformed?

What are you going to do?

• Some bacteria are naturally resistant to antibiotics, but others are not.– How could you use LB/agar plates, some E. coli,

and some ampicillin (an antibiotic) to determine how E. coli cells are affected by ampicillin?

• What would you expect your experimental results to indicate about the effect of ampicillin on the E. coli cells?