Download pGLO Pre-Lab Worksheet- DUE MONDAY 4/24/17

pGLO Bacterial Transformation Lab
Honors Genetics/Ms. Day
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pGLO Bacterial Transformation Lab
Honors Genetics/Ms. Day
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pGLO Bacterial Transformation Lab
Honors Genetics/Ms. Day
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pGLO Bacterial Transformation Lab
Honors Genetics/Ms. Day
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pGLO Bacterial Transformation Lab
Honors Genetics/Ms. Day
WHAT DOES THE pGLO PLASMID LOOK LIKE?
ampr gene
r
(amp = ampicillin resistance)
GFP gene
Plasmid DNA
WHY DO WE USE THE CALCIUM CHOLRIDE?
To get the DNA into the bacteria, we have to poke holes in them with the chemical calcium chloride (CaCl2). CaCl2 will
dissociate into Ca2+ and 2 Cl-, and the positive charge of the Ca2+ cancels the negative charge of the DNA, allowing it to cross
the cell wall and cell membrane. The holes poked to allow the DNA in leaves the bacteria leaky. If we don't keep them on ice,
they'll 'bleed' to death.
WHY DO WE USE HEAT SHOCKING?
Heating the bacteria helps the holes in the membrane and cell wall seal shut. It's like giving the bacteria a fever, so they start to
heal themselves. It's called heat shock.
WHY DO WE USE THE LURIA BROTH?
The LB (Luria-Bertani) broth is both food and water for the bacteria. It will help make the bacteria healthy after poking holes
in them, shoving DNA into them, and giving them a 'fever' to help them heal.
WHAT SPECIMEN ARE WE USING IN LAB?
These bacteria are E. coli, which grow in human intestine. Because they grow in humans, they will grow best at human body
temperature (37°C). 37°C = 98.6°F (normal human body temperature)
HOW WELL DID WE TRANSFORM OUR E.COLI BACTERIA?
Your next task in this investigation is to learn how to determine how well you genetically transformed E. coli cells. This
quantitative measurement is referred to as the transformation efficiency. In many experiments, it is important to genetically
transform as many cells as possible. For example, in some types of gene therapy, cells are collected from the patient,
transformed in the laboratory, and then put back into the patient. The more cells that are transformed to produce a needed
protein, the more likely the therapy will work. The transformation efficiency is calculated to help scientists determine how
well the transformation is working. Because transformation is limited to only those cells that are competent, increasing the
amount of plasmid used does not necessarily increase the probability that a cell will be transformed.
How do you Calculate Transformation Efficiency?
 You are about to calculate the transformation efficiency, which gives you an indication of how effective you were in
getting DNA molecules into bacterial cells.
 Transformation efficiency is a number. It represents the total number of bacterial cells that express the resistance
protein, divided by the amount of DNA used in the experiment.
o
It tells us the total number of bacterial cells transformed by one microgram of DNA.
 The transformation efficiency is calculated using the following formula:
o
Transformation efficiency = Total number of cells growing on the agar plate
Amount of DNA spread on the agar plate (in µg)
 Therefore, before you can calculate the efficiency of your transformation, you will need two pieces of information:
(1) The total number of ampicillin colonies growing on your LB/ +amp plate.
(2) The total amount of ampilcillin plasmid DNA in the bacterial cells spread on the LB/ +amp plate.
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pGLO Bacterial Transformation Lab
Honors Genetics/Ms. Day
Why is it important to have/make a positive control Petri dish (plate)?
 You must make sure that the E.coli can grow in normal conditions. In other words, you must test to make sure that the
bacteria you will be spreading on each plate is healthy and able to grow in conditions that lack antibiotics such as
ampicillin.
Why is it important to have/make a negative control Petri dish (plate)?
 You must make sure that the ampicillin used in the lab is not expired. In other words, you need to make sure the
ampicillin will kill bacteria that lacks the pGLO plasmid containing an ampicillin resistant gene.
MATERIALS
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pGLO Bacterial Transformation Lab
Honors Genetics/Ms. Day
PROCEDURE
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pGLO Bacterial Transformation Lab
Honors Genetics/Ms. Day
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pGLO Bacterial Transformation Lab
Honors Genetics/Ms. Day
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pGLO Bacterial Transformation Lab
Honors Genetics/Ms. Day
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pGLO Bacterial Transformation Lab
Honors Genetics/Ms. Day
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pGLO Bacterial Transformation Lab
Honors Genetics/Ms. Day
Name: __________________________________________________________________ Date: _______________________ Period: _____________
PRE-LAB QUESTIONS: Watch the following youtube.com videos, read the pGLO lab manual provided to your by Ms.
Day then answer the questions.
https://www.youtube.com/watch?v=CMIdyMDHd78 (watch up to 2:52 min)
https://www.youtube.com/watch?v=OZyFX9megs8 (watch up to 5:11 min)
1.
What is the exact name for the glowing gene in the pGLO plasmid, which will be used in lab? __________________________________
2.
Where in nature would you find the pGLO gene being used in this transformation lab activity? ________________________________
3.
What is the difference between “lawn” and “colony” bacterial growth?
4.
Describe the 2 steps (techniques) necessary in getting the pGLO plasmid into the competent bacterium cell via
transformation.
a.
b.
5.
In order for the transformed bacteria to grow and express the GFP gene, what conditions must be meet (i.e.-surround
the bacterial cells)?
a.
b.
6.
What 2 genes are located in the pGLO plasmid? _______________________________________________ & ______________________________
7.
Which part of the pGLO plasmid do you think is the “selective marker” in this lab? ______________________________________________
8.
Why does the bacterial cell need arabinose to express the glowing gene called GFP?
9.
Why do we use the CaCl2 (calcium chloride) in this lab procedure to the make the cell competent? BE SPECIFIC!!!
10. Why do we use the “Luria broth” in this lab procedure?
11. How do you “heat shock” your bacteria in this lab?
12. Why do we use the “heat shocking” in this lab procedure?
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pGLO Bacterial Transformation Lab
Honors Genetics/Ms. Day
13. What specimen are we using in this lab? BE SPECIFIC!!! ________________________________________________
14. What is meant by “transformation efficiency”?
15. What does the following labels (being used in the lab’s procedure) mean?
a.
+pGLO means ______________________________________________________________________________________
b.
-pGLO means ______________________________________________________________________________________
16. You will be using 4 different Petri dishes in this lab. Fill in the table below.
Petri Dish Label
Does this dish
contain the
antibiotic called
ampicillin?
Y or N
Does this dish
contain the sugar
called arabinose?
Y or N
Hypothesis: Will the
bacteria grow on
the dish?
Y or N
Hypothesis: Will the
bacteria GLOW green
on the dish?
Y or N
+pGLO
LB/amp
+pGLO
LB/amp/ara
-pGLO
LB/amp
-pGLO
LB
14. Why do you need to have a positive control plate?
15. Why do you need to have a negative control plate?
16. Which two Petri dishes (plates) are the:
a.
test (experimental) groups ______________________________________________________________________________________
b.
–control groups __________________________________________________________________________________________________
17. Which plates (Petri dishes) should be compared to determine if any genetic transformation has occurred? Why? EXPLAIN
YOUR ANSWER!
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