1. Biotechnology

2. Fluorescent Protein BACTERIAL TRANSFORMATION
This is a plate that has special E.coli bacteria growing on it. These bacteria (that are usually brown) have been tricked into fluorescing different colors.
How did we get them to glow?
That is what we are going to learn how to do today.
But first, we are going to talk about how certain organisms are naturally able to glow, all on their own. 2

3. Bioluminescence vs. Fluorescence
Scorpion- UV Light
Scorpion- Natural Light
Natural Light
Bioluminescence is when an organism is able to produce it's own light, or glow. These mushrooms look normal under natural light, but when they are put in the dark, they can produce their own green glowing light. This is what deep sea animals are able to do. There is no light in the deep sea, so they must create their own light.
Fluorescence is when a organism pulls in one wavelength of light, changes it, and emits a lower wavelength of light that creates the “glow”. This scorpion looks normal under natural light, but when placed under a black light (UV light) the scorpion emits fluorescent light.
In the Dark
A fluorescent organism absorbs light at one wavelength (UV) and a re- emits the light at a visible wavelength= color
Bioluminescent organism produces its own light. 3

4. Many organisms have the ability to fluoresce
Mushroom coral
There are lots and lots of organisms found in nature that can fluoresce all on their own. Here are some examples:
Amphipods
Coral
Jellyfish
Some animals, like this spider in the lower right hand corner, even use fluorescence to attract their mates.
Jellyfish
Amphipod
Spider’s palps 4

5. Jellyfish- Bioluminescence and Fluorescene
You may want to pause before this slide, and ask if any of the students can tell you the difference between bioluminescence and fluorescence.
How can the jellyfish fluoresce, when it is all the way underwater where it can't absorb any light? Remember that fluorescence is when you take in one wavelength of light, change it, and emit another wavelength of light. This jellyfish is able to fluoresce because first it bioluminesces to create blue light. The blue light then goes to GFP(green fluorescent protein), which turns it into green light that we can see. The picture on the right shows a jellyfish in the dark, and all you can see is the outer ring of its body, that fluoresces green.

6. Aequorea victoria and Discovery of GFP-1960’s
The part of the jellyfish that allows it to fluoresce, the GFP that we just saw, was first discovered by a Japanese scientist named Osamu Shimomura. He was able to take the jellyfish that fluoresces in nature, and figure out exactly what protein was responsible for the fluorescence. The picture on the right, is green fluorescent protein. This is the protein that we just saw was responsible for the jellyfish's ability to fluoresce. Osamu Shimomura won the nobel prize, for first discovering, and then isolating this protein.
OSAMU SHIMOMURA Co-winner of Nobel Prize 6

7. Fluorescent Proteins-Applications
Transgenic Fish
Why was discovering this fluorescent protein, so important?
Scientists are now able to take the gene that codes for GFP, and stick it into many other types of cells and organisms. Once they have the gene that codes for GFP, they can express that gene, and make GFP themselves. This is like turning on a light inside the cell so we can study the processes while they are happening.
Here are a few examples:
Neuron- the types of cells that are in your nervous system
Mice- skin cells that fluoresce
Zebra fish- expressing GFP gene
Florescent proteins are revolutionary in bio-technology research.
Transgenic Mice
Neuron 7

8. Roger Tsien and Rainbow Proteins
DsRed.T1
Dimer 2
mRFP1
mgrape 1
mHoneydew
mBanana
mOrange
mTangerine
mStrawberry
mCherry
17 Mut
33 Mut
6 Mut
8 Mut
3 Mut
7 Mut
4 Mut
Roger Tsien is a researcher at UCSD, who was a co-winner of the Nobel Prize (with Osamu Shimomura) for taking GFP, and a few other fluorescent proteins found in nature, and making an entire rainbow of fluorescent protein colors.
Each color was created from making changes (mutating) the DNA sequence.
This slide summarizes about 6 years of research in the lab.

9. E. coli
The type of bacteria we are going to be using today is called E. coli. Some strains of E. coli can make you very sick. However, we will be using lab strain which is harmless but should still be treated with care. Normal E.coli are brown and ugly, but we are going to be making the E. coli fluoresce different colors. How are we going to do that? 9

10. Central Dogma DNA---> mRNA---> Protein---> Trait
We are going to take the normal bacteria, and insert into them, the gene the codes for the fluorescent protein that we were just talking about. The central dogma starts with DNA in the nucleus. The DNA is than transcribed into mRNA, which leaves the nucleus and goes to the ribosome where the protein is made. Once the protein is made, the organism can express the trait.
Today, we are going to be giving the E .coli and extra piece of DNA. The DNA that we are giving the E. coli carries the gene that codes for the fluorescent protein. Once we put the gene into the bacteria, it does the rest. It take the gene we give it, and turns it into the fluorescent protein, through the central dogma. Once the protein is made, the E. coli will start to exhibit the trait, which in this case is fluorescence. 10

11. What is a plasmid? A small circular piece of DNA Naturally occurring
Plasmid Mixes
PM1 PM2
Green Cherry
Blue Tangerine
Grape Yellow
A small circular piece of DNA
Naturally occurring
Can be altered in lab to express protein of interest
Origin
AmpR
Grape-FP
Stop
promoter
Origin
AmpR
BFP
Stop
promoter
Origin
AmpR
GFP
Stop
promoter
A plasmid is a piece of circular DNA that occurs naturally in bacteria. The picture on the left shows a bacterial cell. It has it's own large chromosome, and its own plasmid. Both the chromosome and the plasmid carry genes. The bacteria using the instructions from these genes to make proteins, and express traits. The picture on the right shows the special plasmid that has been made to carry the fluorescent protein gene. The whole point of this lab is to take the normal E.coli, and transform their DNA to include this special plasmid so that the bacteria can make the fluorescent proteins, and glow.

12. How are plasmids engineered?
DNA Plasmid Vector
Host DNA fragments
(i.e. coral or jellyfish FP coding DNA)
Ligate (paste) fragments into cut DNA vector
Cut plasmids open with restriction enzymes +
Cut genomic DNA into fragments
Ask class: What is the special gene that we want to be present on the plasmid?
We want our plasmid to carry the gene that codes for the fluorescent protein. That way, once the gene (on the plasmid) is inside the bacteria, it can turn that gene into the fluorescent protein, and the bacteria will glow.
The first step in getting the gene for fluorescence onto the plasmid is taking the DNA for the organism that is fluorescent on its own, in this case the jellyfish, and cutting it into pieces.
Second, a bacterial plasmid has to be cut open at just the right spot. The last step is to stick the piece of DNA that codes for the fluorescent protein, into the plasmid.
In this lab, we will be using two differing plasmid mixes that have already been made for us. Plasmid mix 1 (PM1) contains the plasmids with the genes that code for green, blue, and grape fluorescent proteins. Plasmid mix 2 (PM2) contains the plasmids with the genes that code for cherry, tangerine, and yellow. Each group will either get a tube labeled PM1 or a tube labeled PM2. These tubes contain your plasmids. The point of this lab is to try to get your bacteria to let in the plasmid, and express the genes for fluorescence that are on the plasmids.
From GFP: From RFP:
PM1 PM2
Green Cherry
Blue Tangerine
Grape Yellow
End result: Plasmid containing FP gene 12

13. What is Transformation?
Bacterial chromosome
Uptake of foreign DNA, often a circular plasmid
Plasmid
Transformation means change. Bacterial DNA transformation means that we are changing the DNA of the bacteria. We will change the DNA of the bacteria to include the plasmid we have engineered. 13

14. What is Transformation?
Bacterial chromosome
Uptake of foreign DNA, often a circular plasmid
Plasmid
Allow bacteria to grow for 1-3 days on plate with ampicillin.
Bacterial chromosome
After we have transformed the bacteria to include our special plasmid, we will let the plates incubate for several days. This will give the bacteria a chance to grow and start expressing the fluorescent protein gene. 14

15. What is Transformation?
Bacterial chromosome
Uptake of foreign DNA, often a circular plasmid
Plasmid
Allow bacteria to grow for 1-3 days on plate with ampicillin.
Bacterial chromosome
After incubation, the plates should have fluorescing bacteria. Each bacterial cell will have created fluorescent proteins.
Bacteria now express cloned fluorescent protein… 15

16. How does the plasmid get in?
CaCl2
Positive charge of Ca++ ions shields negative charge of DNA phosphates
Ca++ O
CH2 P
Base OH
Sugar
The first step is the to take the bacteria cells and mix them with calcium chloride. The calcium chloride is positively charged, and the DNA is negatively charged. The positive charge of the calcium chloride is attracted to the negative charge of the DNA. This makes the positive calcium chloride shield the negative charges of the DNA.
We want to shield the negative charges of the DNA because we do not want the cell membrane of the bacteria to repel the negative charges on the plasmid (DNA). With the negative charges on the DNA shielded, it is easier for the plasmid DNA to get in the bacterial cell.

17. How does the plasmid get in?
Incubate on ice slows fluid cell membrane
Heat-shock increases permeability of membranes
Leave in heat 45 seconds!!!
Too short, and bacteria won't let in plasmid.
Too long, and the bacteria will die.
The second step is called heat shock. The first step of heat shock is to incubate the mixture of plasmid and bacterial cells on ice for 10 minutes. The cold environment of the ice will cause the plasma membrane, which is semi-fluid and moving, to slow down. Once the membrane is cold and slow, we put the mix of bacteria and plasmid into the 420C water for 45 seconds. This shocks the bacterial cell membrane into opening up and letting the plasmid in. After that, we return the bacteria to the ice so that the cell membrane will reclose before the plasmid has a chance to escape.
It is very important that you only leave the mix of bacteria and plasmid in the 42ᵒC water for 45 seconds exactly. If you leave the bacteria in the warmth for too short, they won’t let up the plasmid. If you leave the bacteria in the warmth for too long, they will die.

18. Antibiotic Resistance
promoter
Ampicillin kills any normal bacteria
Any bacteria that are transformed with the plasmid will live on the plates with ampicillin.
They will have the resistance to ampicillin
GFP
Origin
Stop
AmpR
Ask Class: What are antibiotics? What do they do?
Antibiotics are what the doctor gives you when you have a bacterial infection because they kill the bacteria by breaking apart their cell walls.
We will be using the antibiotic called ampicillin. Two of the plates that we will be using have ampicillin mixed in with the LB agar.
The plasmid that we are using to transform the bacteria, carries not only the gene for the fluorescent protein, but also carries the gene for ampicillin resistance. This means that any bacteria that are transformed with our plasmid will be resistant to the ampicillin on the plate.
Ampicillin will kill any normal bacteria that have not taken up the plasmid. Any bacteria that take up the plasmid, will live, even in the presence of ampicillin. The only bacteria that will still live, are ones that have taken up the plasmid

19. FP transformation procedure
Why are your tubes labeled with either a “+” or “-”? The plus means those bacteria were given the plasmid. The “-” tube means those bacteria were not exposed to our plasmid.
Let’s go over the expected results of this lab.
You will have three plates with results: two are control plates and one is your experimental plate.
What do you think these plates will look like?
LB only (-): This plate does not have ampicillin or DNA – what do you think it will look like?
It will have a “lawn” (layer of bacterial growth)
This plate is a control for which factor? – it’s testing that our bacterial cells are healthy.
LB/amp (-): This plate has ampicillin and no DNA – what do you think it will look like?
It should not have any growth at all. – it’s testing that our ampicillin is working. There are no transformed bacteria on the plate, so it should kill any bacteria that try to grow.
LB/amp (+): This plate has ampicillin and the DNA – what do you expect this plate to look like?
It should only have glowing bacteria and no other growth. The ampicillin will kill any other random bacteria and the only bacteria that can grow on the plate are our “super” bacteria that have been transformed to glow and are resistant to the ampicillin. 19