Introduction to pGFP labBacteria Transformation

Old Central Dogma of Molecular Biology

DNA RNA Protein Trait

Uptake of foreign DNA, often a circular DNA called a plasmid

What is transformation?

Bacterial chromosomal DNA

GFP

Amp Resistance

pGFP plasmids

What is a plasmid?

• A plasmid is a small circular piece of DNA (about 2,000 to 10,000 base pairs) that contains important genetic information for the growth of bacteria.

• In nature, this information is often a gene that codes for a protein that will make the bacteria resistant to an antibiotic.

•Bacteria can exchange plasmids with one another.

How do scientists use plasmids?• A plasmid containing resistance to an antibiotic

(usually ampicillin) is used as a vector. The gene of interest is inserted into the vector plasmid and this newly constructed plasmid is then put into E. coli that are sensitive to ampicillin.

• Transformed bacteria can be used to make insulin, human growth hormone, and clotting factor cheaply and in great abundance.

• Vector - Something that is used to transfer something else (a mosquito is a vector for the organism that causes malaria)

How scientists use plasmids (continued)

• The bacteria are then spread over an agar plate that contains ampicillin. The ampicillin provides a selective pressure because only bacteria that have acquired the plasmid can grow on the plate. Therefore, as long as you grow the bacteria in ampicillin, it will need the plasmid to survive and it will continually replicate it, along with your gene of interest that has been inserted to the plasmid.

• Selective Pressure - The same as in evolution - only the organisms that have a particular trait (in this case antibiotic resistance) will survive.

Glowing in Nature

• Many species have the ability to glow

• Most are marine: jellyfish, dinoflagellates

• Some live on land: firefly, glow worm

• Purposes of glowing:Spook predatorsLure preyAttract matesCommunicate

Fireflies

Glow worm and glow worm cave

Why insert glowing genes?

• GFP is a green fluorescent protein that normally is found in jellyfish

• In 1987 Douglas Prasher thought that GFP from a jellyfish could be used to report when a protein was being made in a cell.

• Proteins are extremely small and cannot be seen, even under an electron microscope.

• However if one could somehow link GFP to a specific protein, for example hemoglobin, one would be able to see the green fluorescence of the GFP that is attached to the hemoglobin. It would be a bit like attaching a light bulb to the hemoglobin molecule.

Brainbow miceIn the Brainbow mice, the Harvard researchers have introduced genetic machinery that randomly mixes green, cyan and yellow fluorescent proteins in individual neurons thereby creating a palette of ninety distinctive hues and colors. This is a photograph of the cerebral cortex. It's hard to believe but in non-living preserved brains the outer layers of this portion of the brain are gray, which is why the brain is sometimes called "gray matter". (Confocal image by Tamily Weissman. Mouse by Jean Livet and Ryan Draft.)

Mouse under blue light (left) Same mouse under normal light (right)

Mouse blood vessels (green-GFP) in tumor (red-DsRed). Mouse with brain tumor expressing DsRed.

Malaria is the world's most common and deadly parasitic disease. The World Health Organization estimates that each year 300-500 million cases of malaria occur and more than 1 million people die of malaria. A possible breakthrough in curtailing the spread of malaria carrying mosquitoes was reported in October 2005 the creation of mosquitoes with green fluorescent testicles. Now male mosquito larvae (see picture above) can easily be separated from female mosquito larvae. Without green fluorescent gonads it is impossible to separate mosquito larvae based on their sex, and it is very difficult to separate the adults since they fly about and bite (actually only the females bite).

                                                     

Three 60 day old kittens. Two have been genetically modified to make red fluorescent protein. All three look similar under normal light, but when irradiated with blue light only the two genetically modified kittens glow red. (Photo courtesy of Biology of Reproduction)

Alba – bunny created for “art”

•http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html

Are we going to make bunnies glow green? No, just bacteria.

• We are going to “transform” bacteria by making them take up a commercially prepared plasmid that contains two genes of interest amp and GFP.

• Genetically modified organisms are “transgenic”

amp

GFP

Genes of interest: amp, GFP

• amp – this gene will give our transgenic bacteria resistance to the antibiotic ampicillin

• GFP – this gene will make the bacteria green under regular, white, light and will glow fluorescent green under UV light

Explanation of agar plates

• E. coli starter plate• This plate has the

bacteria we will use in the lab growing in a luria broth (LB) agar plate.

• These bacteria are normal (have NOT been transformed)

Explanation of agar plates• LB/amp/+DNA

• This plate will have E. coli bacteria on LB agar to which ampicillin has been added.

• The +DNA means that the bacteria may have been transformed (if your technique is good).

• If they have been transformed, the bacteria will now have a plasmid with an ampicillin resistant site so they will not be killed by the ampicillin that has been added to the agar

Explanation of agar plates

• LB/amp/+DNA• The +DNA transformed

bacteria will have the amp gene that makes them resistant to ampicillin and the GFP gene that makes them green

• The GFP gene in the plasmid is from a jellyfish.

Explanation of agar plates

• LB/amp/-DNA

• These –DNA bacteria have not received the plasmid. This is the negative control.

• They have not been transformed, so they do not have resistance to the ampicillin that is in the agar.

• Do you think you will see any growth on this plate?

Explanation of agar plates

• LB/-DNA

• This is the positive control plate. These –DNA bacteria are not transformed and are in normal luria broth (LB) agar.

• You should expect to see normal bacterial growth in this plate.

• Read the transformation lab procedure and answer the pre-lab questions

Transformation Procedure

• Suspend bacterial colonies in Transformation Solution, CaCl2

• Add pGLO plasmid DNA to +DNA tube• Place tubes on ice• Heat shock at 42oC and place on ice• Incubate with LB broth• Streak plates