1. Biotech Lab
Paper Plasmid with an introduction to using restriction digest and transformation

2. organism’s visible trait controlled by genes
DO Now: Organisms have visible traits called phenotypes. What are some of your visible traits? Be ready to share your favorite phenotype.
Dark brown hair Blue eyes Straight hair Curly hair Blonde hair Long fingers Green eyes Freckles Dark skin color Fair skin color
Phenotype
organism’s visible trait
controlled by genes
determined the by specific DNA sequences

3. Can you change your phenotype?
*Use artificial methods – hair color, color contact lens, skin bronzer
*Environment can change trait like sun lightening hair or amount of oxygen changing amount of red blood cells
*Alter the DNA…biotechnology

4. Meet E. “Ed” coli or just E. coli… What do you remember about bacteria
Meet E. “Ed” coli or just E.coli… What do you remember about bacteria? E. coli is a type of bacteria like M. luteus. The round circles represent colonies of bacteria. There are millions of cells in each colony.
What is E. “Ed” coli’s phenotype?
Now what is E. “Ed” coli’s phenotype?
The next slide tells you how to grow bacteria for study. Just watch the video link.

5. Why are there some white and blue Ed’s?
How to Grow Bacteria Video
Boil the agar and pour into petri dish.
Cool. Apply bacteria to surface of agar.
Let grow and observe.
Why are there some white and blue Ed’s?

6. Normally, E.coli is white and not blue.
A “gene” is what contributes to producing a phenotype. So blue E.coli has a gene that can produce a blue color and the white E.coli do not have the gene.
Normally, E.coli is white and not blue.
Biotechnology made E.coli blue.
The green arrow is the antibiotic resistance gene which is the gene that makes the bacteria not be killed by antibiotics
The blue insert box is the gene that can help the bacteria make the blue color.
The red square is the ori gene used for the
replication of the plasmid DNA

7. Biotech Lab #3 Paper Plasmid The objective of the lab is to use restriction enzymes to create a paper recombinant plasmid, a plasmid with a new gene inserted.
Ultimately, the plasmid will be put back into E.coli bacteria and give them another phenotype. What new gene will be inserted?
The glowing Gene

8. The Luciferase Gene: Click on links to watch some glowing
Bioluminescence and Fire flies
Top Ten Bioluminescent Organisms
Bioluminescence Surfing
Share your research about which organism contain a glowing gene.
Why do you think they have them in nature?

9. The Task
The plasmid will contain DNA from two different organisms. You will use colored paper, scissors and tape to do this. Can you assign a biotech word for each of the materials you will use?
If you are successful, you will have a two colored paper ring and extra pieces of paper.
Be sure to follow the procedure exactly. Show your teacher when complete.

10. Review: Parts of a Bacterial Plasmid
What color is the ori gene?
What color is the Amp R gene?
How many restriction enzymes can cut the plasmid?
Which part of the plasmid should you cut or mutate to stop the plasmid from making a copy of itself?
Name two antibiotics which are ineffective on bacteria with this plasmid?
If EcoRI cut the plasmid, how many linear pieces would be produced?
If HindIII and BamHI were used to cut the plasmid, how fragments would be produced?

11. How to Create a Recombinant Plasmid
Remove plasmid from bacterial cell.
Extract DNA from organism’s cell. Locate the gene of interest (GOI) to be inserted.
Use specific restriction enzyme(s) to open up the plasmid and create sticky ends.
Use restriction enzyme(s) on the organism’s DNA to cut out the GOI. The inserted gene must have sticky ends.
Both plasmid and gene must have complementary sticky ends.
Mix the plasmids and genes together with ligase. This is a ligation technique.
If successful, the plasmid and gene will circularize.
What was the GOI you inserted into the paper plasmid?

12. Study the diagram and then answer the questions.
What is the GOI?
What restriction enzyme is use to cut the DNA samples?
Why can the human and bacterium DNA combine?
What types of DNA are found in the bacterial cell?
What other genes may be found on the plasmid?
Find the recombinant plasmid in the diagram.

13. What do you do with a recombinant plasmid?
Put it back into a bacterial cell. How?
Use a biotech procedure called a transformation.
Treat bacterial cells with a chemical.
Mix the treated cells and the recombinant plasmids together.
Grow bacteria on agar culture plates.
Look for the bacteria which has the phenotype controlled by the plasmid genes.
All three bacteria have been transformed.
Draw a bacterial cell which has not been transformed.
Click on the next slide to see what your bacteria
would look like if they picked up your paper plasmid
with the lux gene.

14. Transformation:
Above is a picture of millions of bacterial cells. They all
Are glowing which means each cell has taken up the plasmid with the lux gene. What would a nonglowing colony of cells tell you about the bacteria?
Watch the Transformation Procedure

15. In the above illustration, find the bacteria with a plasmid and find the bacteria that do not have a plasmid. The ones with the plasmid can be used to clone the inserted gene. Click on the link to understand gene cloning using a recombinant plasmid as a vector. A vector is something that carries something. What is the plasmid carrying into the bacterial cell?
What is Gene Cloning?

16. What is done with clones of cells
What is done with clones of cells? Use the copies of the genes for… Read Sections 12.6, 7, 8

17. Video links to help understand recombinant biotechnology.
DNA Cloning
Glowing Protein Transformation
bozeman Transformation
Learn how insulin is made using recombinant plasmid