Introduction to Transformation

Central Dogma of Molecular Biology DNARNAProteinTrait

Uptake of foreign DNA by a bacterium, often a circular DNA called a plasmid What is transformation? Bacterial chromosomal DNA rfp Amp Resistance 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 through conjugation.

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, sea anemones, dinoflagellates Some live on land: firefly, glow worm Purposes of glowing:  Spook predators  Lure prey  Attract mates  Communicate

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 mice In 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 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”

Glowing fish for sale

Other glowing mammals

We will be using the rfp gene These kitten have had the rfp gene inserted now.org/health/2011/09/doggonn-it-this- kitty-may-be-researchers’-new-best- friend now.org/health/2011/09/doggonn-it-this- kitty-may-be-researchers’-new-best- friend

Are we going to make kittens glow red? No, just bacteria. We are going to “transform” bacteria by making them take up a commercially prepared plasmid that contains three genes of interest, amp r, araC and rfp. Genetically modified organisms are “transgenic”

Genes of interest: amp, araC, GFP amp r – this gene will give our transgenic bacteria resistance to the antibiotic ampicillin araC – this gene will produce a protein in the presence of arabinose (a sugar that is added to agar) that will allow the bacteria to turn on the rfp gene rfp – in the presence of arabinose, this gene will “turn on” and cause the transformed (transgenic) bacteria to glow red

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 plate LB is food for the bacteria Half of this plate will receive transformed bacteria, P+ The other half of this plate will receive un- transformed bacteria, P- Do you expect bacteria to grow on either side of this plate? Why or why not?

Explanation of agar plates LB/amp plate This plate has LB agar to which ampicillin has been added. Half of this plate will receive transformed bacteria, P+ The other half of this plate will receive un- transformed bacteria, P- Do you expect bacteria to grow on either side of this plate? Why or why not?

Explanation of agar plates LB/amp/ara plate This plate will have transformed bacteria, P+ growing on agar that has both ampicillin and arabinose added to it. If your technique is good, you should expect to see red glowing bacteria in this plate.

Transformation Procedure Suspend bacterial colonies in Transformation Solution, CaCl 2 Add pARA-R plasmid DNA to P+ tube Place tubes on ice Heat shock at 42 o C and place on ice Streak plates