Idea Transcript
PGLO TRANSFORMATION LAB (AP LAB 7)
araC ori
pGLO bla
BIO-RAD lab book
GFP
PURPOSE: To observe gene expression in real time by performing a genetic transformation procedure on E. coli bacteria using a plasmid as a vector. If successful, the plasmid will provide the E. coli with two new traits: • expression of a gene that codes for Green Fluorescent Protein (GFP) bioluminescence under the control of an operon • resistance to the antibiotic Ampicillin
BACKGROUND
OLD CENTRAL DOGMA OF MOLECULAR BIOLOGY
WHAT IS TRANSFORMATION?
Uptake of foreign DNA plasmid – that produces new traits in the bacteria
Bacterial chromosomal DNA
pGLO plasmids
WHAT IS A PLASMID?
A plasmid is a small circular piece of DNA (about 2,000 to 10,000 base pairs long) 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.
WHAT IS THE GFP GENE?
GFP is a green fluorescent protein that naturally occurs in some bioluminescent jellyfish.
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 predators Lure prey Attract mates Communicate
Fireflies Glow worm and glow worm cave
WHY DO SCIENTISTS USE PLASMIDS?
A plasmid 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 or some other target. For example: transformed bacteria can be used to make insulin, human growth hormone, and clotting factor cheaply and in great abundance.
araC ori
pGLO bla
GFP
Vector - Something that is used to transfer something else (a mosquito is a vector for the organism that causes malaria)
Jellyfish Gene put into Other Critters
http://www.technologyreview.com/files/21291/monkey_x600.jpg
ALBA – BUNNY CREATED FOR “ART”
•http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html
Three 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)
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.
In Brainbow mice, 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. In non-living preserved brains the outer layers of this portion of the brain are gray. (Confocal image by Tamily Weissman. Mouse by Jean Livet and Ryan Draft.)
REAL-WORLD APPLICATION:
Malaria is the world's most common and deadly parasitic disease.
The World Health Organization estimates that 300-500 million cases of malaria occur each year 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. Without green fluorescent gonads it is impossible to separate mosquito larvae based on their sex, and it is very difficult to separate the adults. Now male mosquito larvae can easily be separated from female mosquito larvae.
ARE WE GOING TO MAKE CATS GLOW GREEN? NO, JUST BACTERIA.
In this lab, you will “transform” bacteria by making them take up a commercially prepared plasmid that contains three genes of interest: amp, araC and GFP Genetically modified organisms are called “transgenic”
BACTERIAL TRANSFORMATION The uptake of plasmid DNA Bacterial Cell Chromosomal DNA
Plasmids
The bacterium Escherichia coli or E. coli is an ideal organism for the molecular geneticist to manipulate and has been used extensively in recombinant DNA research. It is a common inhabitant of the human colon and can easily be grown in suspension culture in a nutrient medium such as Luria broth, or in a petri dish of Luria broth mixed with agar (LB agar) or nutrient agar.
source of GFP
Aequorea victoria Source of “glowing gene” for this experiment
PGLO
PLASMID araC
OriPlasmid Replication ori genes
pGLO
bla
GFP
Arabinose Operon (inducible) Turns on (makes a protein) when arabinose sugar is present Allows bacteria to “turn on” the Fluorescence because it has been linked into the same operon system PBAD arabinose promoter GFP-Green Fluorescent Protein - Glows green in fluorescent light
bla (β-lactamase) - “on” all the time - makes protein that breaks down ampicillin - provides ampicillin resistance
Plasmids can transfer genes that occur naturally within them, or they can act as carriers for introducing foreign DNA from other sources into recipient bacterial cells. Restriction endonucleases can be used to cut and insert pieces of foreign DNA into the plasmid vectors
GENES OF INTEREST: AMP, ARAC, GFP
amp – 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 GFP gene GFP – in the presence of arabinose, this gene will “turn on” and cause the transformed (transgenic) bacteria to glow green
ARABINOSE OPERON REGULATION ara Operon araC
B
A
D Effector (Arabinose)
araC
B
A
D
RNA Polymerase araC
B
A
D
INDICIBLE OPERON: The presence of arabinsoe turns on genes that make enzymes (proteins) to digest the sugar arabinose
PGLO
REGULATION
ara Operon araC
B
A
D
GFP Gene
Effector (Arabinose)
araC
B
A
D
GFP Gene
RNA Polymerase araC
B
A
D
GFP Gene
GFP GENE HAS BEEN ADDED TO ara OPERON WHEN ARABINOSE IS PRESENT, OPERON IS TURNED ON and GFP GENE IS EXPRESSED TOO!
GETTING STARTED
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/
This plate will have E. coli bacteria on LB agar to which ampicillin has been added.
LB/amp/ara This plate will have bacteria growing on agar that has both ampicillin and arabinose added to it.
WHAT SHOULD YOU EXPECT?
If your technique is good, you should expect to see green glowing bacteria in some plates and not others. Read the transformation lab procedure and answer questions 1 – 4 for Lesson 1.
protocol BACTERIAL TRANSFORMATION
MAKE OBSERVATIONS! Be sure to make observation and answer questions on pg. 30 of your packet – before you start the experiment. The liquid (broth) and solid (agar) nutrient media are made from an extract of yeast and an enzymatic digest of meat byproducts, which provide a mixture of carbohydrates, amino acids, nucleotides, salts, and vitamins, as nutrients for bacterial growth. The foundation, agar, is derived from seaweed. It melts when heated, forms a solid gel when cooled and functions to provide a solid support on which to culture bacteria.
Gather Supplies - 10 groups Label Tubes
+ pGLO
- pGLO
What do these represent?
Transformation solution (CaCl2)
Use sterile pipette to add 250µL transformation solution to pGLO + and – tubes
WHEN USING THE PIPETTE FOR MEASUREMENTS TAKE INTO ACCOUNT THE FOLLOWING GRADUATIONS
GET YOUR RACK ON ICE!
INNOCULATE TUBES WITH E. COLI BACTERIA Get new loop Pick one colony Twirl loop in –pGLO tube
Pick one colony Twirl loop in +pGLO tube USE SPECIAL GARBAGE BAG FOR DISPOSAL OF USED LOOPS
EXAMINE PGLO PLASMID DNA
Use UV light to examine pGLO plasmid vial
UV light can be harmful to your eyes! Wear your goggles. Do not shine in eyes.
GFP = Green Fluorescent Protein isolated from jellyfish
USED AS A GENETIC TOOL http://www.mshri.on.ca/nagy/GFP%20mice.jpg
PLASMID DNA TRANSFER THIS STEP IS CRUCIAL! Look closely to make sure you have a film of solution across the ring.
(Similar to soapy film when you blow bubbles) ADD PLASMID TO + TUBE DO NOT ADD PLASMID TO - TUBE
GET YOUR RACK ON ICE!
10 minutes!
WHILE YOUR TUBES COOL LABEL YOUR PLATES
UPSIDE DOWN AND WRITE LABELS ON BOTTOM … NOT ON TOP!
SHOCKING INCREASES UPTAKE OF FOREIGN DNA (PLASMID) OSMOTIC SHOCK =Transforming solution CaCl2
HEAT SHOCK RAPID TEMPERATURE CHANGE is the key
50 SECONDS!!
2 MINUTES
•Place foam rack with + and – tubes on desktop •Use new sterile pipette to add 250 µL LB (broth) to + tube •Use new sterile pipette to add 250 µL LB (broth) to – tube • Incubate at ROOM TEMPERATURE 10 min
TAP WITH FINGER TO MIX! Use NEW STERILE pipette for each vial to add 100 µL bacterial suspension to CORRECT DISH (CHECK LABELS!) Use a NEW STERILE LOOP FOR EACH PLATE to spread suspension evenly on surface of plate
QUICKLY REPLACE LIDS
FLIP PLATES UPSIDE DOWN STACK AND TAPE LABEL WITH YOUR GROUP NAME
PLACE IN INCUBATOR
REASONS FOR EACH TRANSFORMATION STEP Ca++
The transformation solution CaCl2
It is thought that the Ca2+ cation neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane to allow the DNA to enter the cells.
Ca++
O
O P O O CH2
Base
O
Sugar O
Ca++
O P O
Base
O CH2
O
Sugar OH
Reasons for Each Transformation Step
Incubation on ice slows fluid cell membranes
Heat-shock increases permeability of cell membrane
Nutrient broth incubation allows beta lactamase expression
SELECTION FOR PLASMID UPTAKE
Antibiotic becomes a selecting agent only
bacteria with the plasmid will grow on antibiotic (ampicillin) plate all bacteria grow
only transformed bacteria grow
a a a
a
a a
LB plate
a
a a a
a a a
a a
a a
LB/amp plate cloning
Transformation Results LB PLATE Luria Broth +
- PGLO = NO Plasmid
→ All cells grow since there is no antibiotic on the plate
Transformation Results LB/AMP PLATE Luria Broth with antibiotic +
- PGLO = NO plasmid
→ NO GROWTH Cells without plasmid don’t have antibiotic resistance. Can’t grow on media with antibiotic added.
How does ampicillin in the agar act as a “selective pressure”?
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.
Transformation Results LB/AMP PLATE Luria Broth with antibiotic +
+ PGLO = Plasmid added
→ LAWN Cells with plasmid have antibiotic resistance gene so can grow on media with antibiotic
Transformation Results LB/AMP/ARA PLATE Luria Broth + antibiotic| + arabinose
+ + PGLO = Plasmid added
Cells with pGLO plasmid GROW & GLOW -can grow on media with antibiotic GLOW on media with arabinose (turns on GFP gene)
→
the operon
GENE EXPRESSION IN PROKARYOTES (DIFFERENT IN EUKARYOTES)
GENE REGULATION REVIEW
Organisms regulate expression of their genes and ultimately the amounts and kinds of proteins present within their cells for a myriad of reasons.
Gene regulation not only allows for adaptation to differing conditions, but also prevents wasteful overproduction of unneeded proteins which would put the organism at a competitive disadvantage.
The genes involved in the transport and breakdown (catabolism) of food are good examples of highly regulated genes. For example, the sugar arabinose is both a source of energy and a source of carbon.
E. coli bacteria produce three enzymes (proteins) needed to digest arabinose as a food source. The genes which code for these enzymes are not expressed when arabinose is absent, but they are expressed when arabinose is present in their environment.
Regulation of the expression of proteins often occurs at the level of transcription from DNA into RNA. This regulation takes place at a very specific location on the DNA template, called a promoter, where RNA polymerase sits down on the DNA and begins transcription of the gene. In bacteria, groups of related genes are often clustered together and transcribed into RNA from one promoter. These clusters of genes controlled by a single promoter are called operons.
The regulation of gene expression is a complicated and varied process. One, more well understood process is the operon.
An operon is made up of several structural genes3 arranged under a common promoter1 and regulated by a common operator2. operon
1
3 2
It is defined as “a set of adjacent structural genes, plus the adjacent regulatory signals that affect transcription of the structural genes.”
Genes are transcribed together into a mRNA strand and either translated together, or undergo trans-splicing to create several strands of mRNA that each encode a single gene product translated separately. The result of this is that the genes contained in the operon are either expressed together or not at all.
BASICS OF TRANSCRIPTION AND TRANSLATION
Transcription is the synthesis of RNA under the direction of DNA Transcription produces messenger RNA (mRNA) Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA Ribosomes are the sites of translation
THE LAC OPERON The first operon to be described was the lac operon in Escherichia coli.
The Lac operon - showing its genes and its binding sites.
Provides a typical example of operon function. It consists of three adjacent structural genes, a promoter, a terminator, and an operator.
The lac operon is required for the transport and metabolism of lactose in Escherichia coli and some other enteric bacteria. The lac operon is regulated by several factors including availability of glucose and lactose. (This is an example of the negative inducible model.)
In the "repressed" state, the repressor IS bound to the operator. The gene is essentially turned off. There is no lactose to inhibit the repressor, so the repressor binds to the operator, which obstructs the RNA polymerase from binding to the promoter and making lactase.
off
repressor RNA polymerase
promoter
operator gene sequence for lactase production
on
mRNA
polypeptide ribosome
The gene is turned on. Lactose is inhibiting the repressor, allowing the RNA polymerase to bind with the promoter, and express the genes, which synthesize lactase. Eventually, the lactase will digest all of the lactose, until there is none to bind to the repressor. The repressor will then bind to the operator, stopping the manufacture of lactase
Control of an operon is a type of gene regulation that enables organisms to regulate the expression of various genes depending on environmental conditions. Operon regulation can be either negative or positive by induction or repression.
APPLICATIONS IN THE “REAL WORLD”
THE PROMOTER A nucleotide sequence that enables a gene to be transcribed. The promoter is recognized by RNA polymerase, which then initiates transcription. In RNA synthesis, promoters indicate which genes should be used for mRNA creation – and, by extension, control which proteins the cell manufactures.
THE OPERATOR A segment of DNA that a regulator binds to. It is classically defined in the lac operon as a segment between the promoter and the genes of the operon.
THE REPRESSOR In the case of a repressor, the repressor protein physically obstructs the RNA polymerase from transcribing the genes.
ARABINOSE OPERON
The three genes (araB, araA and araD) that code for three digestive enzymes involved in the breakdown of arabinose are clustered together in what is known as the arabinose operon.3 These three proteins are dependent on initiation of transcription from a single promoter, PBAD. Transcription of these three genes requires the simultaneous presence of the DNA template (promoter and operon), RNA polymerase, a DNA binding protein called araC and arabinose. araC binds to the DNA at the binding site for the RNA polymerase (the beginning of the arabinose operon). When arabinose is present in the environment, bacteria take it up. Once inside, the arabinose interacts directly with araC which is bound to the DNA. The interaction causes araC to change its shape which in turn promotes (actually helps) the binding of RNA polymerase and the three genes araB, A and D, are transcribed. Three enzymes are produced, they break down arabinose, and eventually the arabinose runs out. In the absence of arabinose the araC returns to its original shape and transcription is shut off.
PGLO
RECOMBINANT DNA
The DNA code of the pGLO plasmid has been engineered to incorporate aspects of the arabinose operon. Both the promoter (PBAD) and the araC gene are present. However, the genes which code for arabinose catabolism, araB, A and D, have been replaced by the single gene which codes for GFP. Therefore, in the presence of arabinose, araC protein promotes the binding of RNA polymerase and GFP is produced. Cells fluoresce brilliant green as they produce more and more GFP. In the absence of arabinose, araC no longer facilitates the binding of RNA polymerase and the GFP gene is not transcribed. When GFP is not made, bacteria colonies will appear to have a wild-type (natural) phenotype—of white colonies with no fluorescence. This is an excellent example of the central molecular framework of biology in action: DNA➜RNA➜PROTEIN➜TRAIT.
The pGLO plasmid, which contains the GFP gene, also contains the gene for betalactamase, which provides resistance to the antibiotic ampicillin, a member of the penicillin family. Beta-lactarmase inactivates the ampicillin present in the LB nutrient agar to allow bacterial growth. Only transformed bacteria that contain the plasmid and express beta-lactamase can grow on plates that contain ampicillin.
TRANSFERRING BACTERIAL COLONIES FROM AGAR PLATES TO MICROTUBES
The process of scraping a single colony off the starter plate leads to the temptation to get more cells than needed. A single colony that is approximately 1 mm in diameter contains millions of bacterial cells. To increase transformation efficiency, students should select 24 colonies that are 1-1.5 mm in diameter. Selecting more than 4 colonies may decrease transformation efficiency. Select individual colonies rather than a swab of bacteria from the dense portion of the plate, the bacteria must be actively growing for transformation to be successful.
PLASMID DNA TRANSFER
The transfer of plasmid DNA from its stock tube to the transformation suspension is crucial. When dipping the inoculating loop into the container, you must look carefully at the loop to see if there is a film of plasmid solution across the ring, similar to film on wand when blowing bubbles.
HEAT SHOCK
Since the heat shock increases the permeability of the cell membrane to DNA, it is very important to follow the directions regarding time in the warm bath and the rapid temperature change. While the mechanism is not known, the duration of the heat shock is critical and has been optimized for the type of bacteria used and the transformation conditions employed. For optimal results, the tubes containing the cell suspension must be taken directly from ice, placed into the water bath at 42°C for 50 sec and returned immediately to the ice.
RECOVERY
The 10 minutes incubation period following the addition of LB nutrient broth allows the cells to recover and to express the ampicillin resistance protein beta-lactamase so that the transformed cells survive on the ampicillin selection plates.