1. Transformation of E. coli
Biotechnology
Transformation of E. coli
6/9/2018

2. Introductory Question
Explain how you can determine whether a bacterial cell contains the recombinant plasmid.
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3. Essential Questions What is transformation?
How do E.coli cells take up recombinant DNA molecules?
What makes bacteria cells “competent”?
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4. Terminology
Plasmid- Circular DNA found outside of the bacterial genome
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5. Plasmids exist in two forms
Supercoiled- DNA molecule winds around each other to produce condensed entangled molecule
Natural form of the plasmid; form that the bacterium likes to take up
Relaxed – non supercoiled
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6. What is GFP?
Green fluorescent protein- natural protein found in jellyfish that glows green in along wave UV light
Gene encoding GFP was isolated; the gene we will use in the transformation lab
When expose the protein to long wave UV light will fluoresce green
nobelprize.org
BIO-RAD
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7. Green Fluorescent Protein
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8. GFP protein Useful tool in cell biology
Often fused to other proteins to study various biochemical process
Creates a chimeric protein = GFP + protein of interest
Because it is easy to visualize serves as a good biological tag
Can use this biological tag to study protein localization and trafficking within cells.
What organelle is the protein located in within the cell?
How does the protein get there after it is synthesized on the ribosome?
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9. GFP as a biological tag
Mice genetically engineered to express GFP in its skin cells
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10. Normal Light Can you tell how many mice are transformed? 6/9/2018
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11. PROPERTY OF PIMA COUNTY JTED, 2010
Think-Pair-Share
Can you think of an every day example of biological tag?
3. Think-pair-share
teacher presents a question
teacher gives wait time for student to form answer
teacher instructs students to share their answer with a partner
teacher calls on non-volunteers to share with the class
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12. Our Recombinant Plasmid
The recombinant DNA molecule we are going to use in the transformation lab
Our Recombinant Plasmid
The recombinant plasmid contains two genes
1. Gene encoding GFP
2. Gene encoding ampicillin resistance
GFP gene
Ampicillin resistance gene
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13. Terminology
Antibiotic- “against life” medicine that kill bacteria (or prevent them from replicating)
surgeryencyclopedia.com
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14. Why don’t antibiotics work on colds?
Think-Pair-Share
Why don’t antibiotics work on colds?
3. Think-pair-share
teacher presents a question
teacher gives wait time for student to form answer
teacher instructs students to share their answer with a partner
teacher calls on non-volunteers to share with the class
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15. Bacteria Cell Structure
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16. Antibiotic Ampicillin
Derivative of penicillin
Inhibits bacterial growth by interfering with the synthesis of bacterial cell walls
Bacteria cells without the cell wall pop open, killing the bacterium
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17. Ampicillin Resitance Gene
Encodes for the enzyme b-lactamase
Secreted by cells transformed with the recombinant plasmid into surrounding bacterial growth medium and breaks down the ampicillin present
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18. Transformation
Process by which bacterium takes up and expresses exogenous DNA
Happens in nature when bacterium cells die and release their DNA, allowing other bacteria cells to take up the DNA
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19. Transformation
Provides the bacterium with a newly acquired genetic trait that is stable and heritable
For example, can make a bacterium resistant to ampicillin by taking up the antibiotic resistance gene
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20. How many Bacteria Acquire Antibiotic Resistance
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21. Competency
For bacterium to take up DNA they must be in a particular physiological state referred to as competency
When competent the bacterial cell wall is permeable to macromolecules such as DNA
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22. E.coli
The bacterium used in the transformation lab does not naturally enter a state of competency
Must artificially make them competent
Accomplished by exposing E.coli
With chloride salts of metal cations, commonly CaCl2
Exposing to abrupt temperature change
Heat shock at 42oC for 90 sec followed by putting them on ice
These treatments affect the structure and permeability of the cell wall allowing them to temporarily take up DNA
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23. Transformation efficiency
Transformation not 100% efficient
Approximately 1 in 10,000 cells successfully incorporates exogenous DNA
However, because there are so many cells in a sample (approx. 1 x 109) can typically obtain visible colonies on selective (with antibiotic) media
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24. Answer the following questions IYOW
What is transformation?
Recombinant DNA often contains the ampicillin resistant gene. What does the ampicillin resistant gene encode for? How does that protein work?
What is transformation considered not very efficient?
How are E.coli cells made competent to take up DNA?
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25. Transformation Lab Part I
Read through introduction to transformation in lab manual and answer focus questions in lesson 1 in your lab notebook
After completing focus questions, read the procedures in Lesson 2 and complete your Pre-Lab write up
Your Pre-Lab should include
Hypothesis
Experimental Design- Experimental treatment, control, and fixed variables
Summary of procedures
Get instructor’s signature upon completion
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