Download BACTERIAL TRANSFORMATION Lab 15

BACTERIAL TRANSFORMATION
Lab
15
The most important discoveries of the laws, methods, and progress of Nature nearly always spring from the
examination of the smallest objects which she contains.
Jean Baptiste de Lamarck, Philophie Zoologique
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(1809)
CAUTIONS AND PITFALLS
Disinfect your work area before and after working with bacterial cultures. Many
microorganisms thrive under the same conditions as the harmless E. coli strain being used in this
experiment, therefore it is important to maintain sterile conditions to minimize the possibility of
contamination with foreign bacteria or fungi.
Sterile technique will be used throughout this experiment. Keep in mind that everything that
comes in contact with E. coli bacteria has been, or must be, pre-sterilized including: calcium
chloride solution, Luria broth, transfer pipettes, inoculating loops, spreaders, culture tubes, and
petri plates.
Always open wrappers of sterile transfer pipettes and loops so that you do not touch the tip
end of the pipet or loop. Open at the opposite end.
Do not reuse pipettes or transfer loops.
Dispose of all contaminated materials in a container of appropriate disinfectant. Do not lay
pipettes or loops on the table top.
Wash hands before leaving the laboratory.
OBJECTIVES
After completing this lab you should be able to:
1. Explain how a genotypic change can affect the phenotype of an organism.
2. Describe cell membrane permeability and discuss how it can be manipulated in bacterial
transformation.
3. Predict the results of a controlled transformation experiment using antibiotic resistance plasmids.
4. Demonstrate sterile technique for handling nonpathogenic as well as pathogenic microorganisms.
INTRODUCTION
Transformation means "change". In molecular biology, transformation refers to a form of genetic
change in which bacteria take up free DNA from the environment enabling them to express a new
phenotype. The naturally occurring phenomenon of bacterial transformation has been of critical
importance in the development of molecular biology. Today it is one of the most widely used and
versatile techniques in genetic engineering.
Most bacteria do not "transform" readily, but researchers have discovered ways to enhance the uptake
of DNA so that this naturally occurring means of genetic alteration can take place more efficiently. In
1970, it was discovered that certain conditions can help bacterial cells
absorb exogenous (free) DNA. Cells treated in this manner have been made "competent" to take up
exogenous DNA, significantly increasing their transformation efficiency. These conditions include:
* Working with rapidly growing cells
* Suspending cells in cold calcium chloride
* Heat-shocking the cold cells at 40-42o C.
* Using intact plasmids rather than DNA fragments
In this experiment we will attempt to transform E. coli with a plasmid, named pGal. This plasmid,
developed at UC Berkeley, is one of the more common plasmids used in recombinant DNA protocols.
The strain of E. coli (JM101) used in this experiment is non-pathogenic, non-infectious and can easily
be rendered competent by the methods described. Some strains of E. coli are normal inhabitants of
the human digestive tract, while other pathogenic strains have been known to cause Traveler's Diarrhea
a.k.a. Montezuma's Revenge. The transforming plasmid (pGal), carries genes coding for two
identifiable phenotypes:
* The ampr gene codes for resistance to the antibiotic ampicillin
* The lacz gene codes for the enzyme β-galactosidase
Recall that in recombinant DNA protocols, plasmids are often used as vectors or "vehicles" that
transport genetic material into and out of cells. Plasmid vectors used in research are frequently
engineered to contain DNA for both a desired trait (such as the gene for a rDNA hormone or enzyme)
and an identifying "marker" trait for selecting organisms that have taken up the desired gene. pGal is a
popular plasmid vector because it carries two such markers. Ampicillin is an antibiotic that prevents E.
coli from constructing cell walls. Beta-galactosidase is an enzyme that catalyzes the breakdown of
lactose (milk sugar). In this experimental protocol, a chemical analog named X-gal will be added to the
growth media instead of lactose. The significance of this alternate substrate is that X-gal produces a
visible blue product when exposed to the beta-galactosidase enzyme. Wild-type E. coli strain JM101
bacteria lack both ampicillin and beta-galactosidase genes. Thus we will be able to select
transformed colonies based on their ability to grow on ampicillin and their ability to produce
blue colonies in the presence of X-gal.
1. Name the bacteria used in this experiment
2. Name the plasmid vector used in this experiment
3. Transformed E coli cells will express two new phenotypes, what are they:
RESULTS
Results can be observed in 24-48 hours. Your lab report will consist of answers to the post lab
questions plus an informal oral report to the class.
Each lab group will be assigned a specific part of the experiment to present to the class.
All lab partners are expected to contribute to the presentation.
Lab Group Presentation Topics
Sterile Technique
Bacterial Transformation
pGal Plasmid
Experimental Results
NAME
SECTION
BACTERIAL TRANSFORMATION POST LAB QUESTIONS
Answer these questions as completely as possible. Turn in the questions in place of a formal lab report.
1. Lightly shade the circles to indicate the plates you predict will show a confluent bacterial lawn. Use
clear or colored circles to indicated colonies on the plates where only some growth is expected.
2. Compare your predicted results with the actual results after incubation. Explain why growth or no
growth occurred for each plate.
a)
E. coli not exposed to pGal grown on nutrient LB agar.
b) E. coli exposed to pGal grown on nutrient LB agar:
LB
(-)
LB
(+)
c) E. coli not exposed to pGal grown on plate with AMP:
LB
AMP
(-)
d) E. coli exposed to pGal grown on plate with AMP:
LB
AMP
(+)
e) E. coli not exposed to pGal grown on plate with AMP and X-gal:
f) E. coli exposed to pGal grown on plate with AMP and X-gal:
LB
AMP
X-Gal
(-)
LB
AMP
X-Gal
(+)