1. Introduction to Biotechnology Transformation and more!
Do you know how a car engine works?
4-stage cylinder?
Introduction to Biotechnology Transformation and more!
Chapter 20
Section Pgs
Objective: I can describe several different types of biotechnology, such as plasmid cloning and transformation, in order to best determine how to cut a gene of interest out and insert it into a plasmid.

2. Prokaryotic DNA Packaging (Review)
1 circular bacterial chromosome
NOT a plasmid…
Plasmid can refer to small(er) circular molecules of DNA besides “main” chromosome
Self-Replicating, so will pass on
Confusing, because plasmid can be incorporated into “main” or stay isolated
Prokaryotic Cell Division is called…
Binary Fission, which is a type of…
Asexual Reproduction

3. Prokaryotic Sexual “Reproduction”
Conjugation
Uses pili (singular pilus)
Transfers DNA
Not reproduction recombination
Can pass plasmid(s) or part of “main”
Can either remain as plasmid in receiver
Can be incorporated into main bacterial chromosome

5. Conjugation: A Natural Transformation
Bacteria naturally transfer genetic info (in certain conditions)  helps survival (adaptation + evolution)
Also viruses can transform genetic makeup of host…
Similar to whose experiment…?
Griffith’s Experiment

6. Can we artificially transfer genes?
If bacteria can naturally take in foreign DNA (undergo transformation), can we artificially make them take up DNA we want them to?
Yes: Biotechnology (DNA Technology)
Genetic Engineering  Recombinant DNA
What’s the point?
Allow for gene cloning (just gene)
Allows for GMO (Genetically Modified Organisms)
How? (specifically)?

7. Bacteria of Choice
E.coli Escherichia coli Use bacteria as “factory” Can clone a gene (DNA) by letting E.coli reproduce (divide)

8. A cell with DNA Plasmid containing the gene of interest E. coli
bacterium 2
The cell’s DNA
is isolated.
Bacterial
chromosome 1
A plasmid
is isolated.
Gene of
interest
DNA
Figure 12.1B_s1 An overview of gene cloning (part 1, step 1) 8

9. A cell with DNA Plasmid containing the gene of interest E. coli
Figure 12.1B_s2
A cell with DNA
containing the gene
of interest
Plasmid
E. coli
bacterium 2
The cell’s DNA
is isolated.
Bacterial
chromosome 1
A plasmid
is isolated.
Gene of
interest
DNA 3
The plasmid is cut
with an enzyme. 4
The cell’s DNA is cut
with the same enzyme.
Gene
of interest
Figure 12.1B_s2 An overview of gene cloning (part 1, step 2) 9

10. A cell with DNA Plasmid containing the gene of interest E. coli
Figure 12.1B_s3
A cell with DNA
containing the gene
of interest
Plasmid
E. coli
bacterium 2
The cell’s DNA
is isolated.
Bacterial
chromosome 1
A plasmid
is isolated.
Gene of
interest
DNA 3
The plasmid is cut
with an enzyme. 4
The cell’s DNA is cut
with the same enzyme.
Gene
of interest 5
The targeted fragment
and plasmid DNA
are combined.
Figure 12.1B_s3 An overview of gene cloning (part 1, step 3) 10

11. A cell with DNA Plasmid containing the gene of interest E. coli
Figure 12.1B_s4
A cell with DNA
containing the gene
of interest
Plasmid
E. coli
bacterium 2
The cell’s DNA
is isolated.
Bacterial
chromosome 1
A plasmid
is isolated.
Gene of
interest
DNA 3
The plasmid is cut
with an enzyme. 4
The cell’s DNA is cut
with the same enzyme.
Gene
of interest 5
The targeted fragment
and plasmid DNA
are combined.
Figure 12.1B_s4 An overview of gene cloning (part 1, step 4) 6
DNA ligase is added,
which joins the two
DNA molecules.
Recombinant
DNA
plasmid
Gene
of interest 11

12. The recombinant plasmid is taken up by a bacterium
Figure 12.1B_s5
Recombinant
DNA
plasmid
Gene
of interest 7
The recombinant plasmid
is taken up by a bacterium
through transformation.
Recombinant
bacterium
Figure 12.1B_s5 An overview of gene cloning (part 2, step 1) 12

13. The recombinant plasmid is taken up by a bacterium
Figure 12.1B_s6
Recombinant
DNA
plasmid
Gene
of interest 7
The recombinant plasmid
is taken up by a bacterium
through transformation.
Recombinant
bacterium 8
The bacterium
reproduces.
Figure 12.1B_s6 An overview of gene cloning (part 2, step 2)
Clone
of cells 13

14. The recombinant plasmid is taken up by a bacterium
Figure 12.1B_s7
Genes may be inserted
into other organisms.
Recombinant
DNA
plasmid
Gene
of interest 7
The recombinant plasmid
is taken up by a bacterium
through transformation. 9
Recombinant
bacterium
Harvested
proteins
may be
used
directly. 8
The bacterium
reproduces.
Figure 12.1B_s7 An overview of gene cloning (part 2, step 3)
Clone
of cells 14

15. animation file

16. How can cut gene of interest out? How can cut plasmid to insert?
Restriction Enzymes: natural defense (a.k.a. restriction endonucleases)
Enzymes that recognize a particular sequence in DNA and cut there
Sequence must be “symmetrical” in that it is a “complementary palindrome”
Area to be cut = restriction site
animation file

17. Restriction Enzymes Details
Usually cut in a staggered format
Results in “sticky ends”
Allows to hydrogen bond to other restriction fragments
Fragments of DNA cut by restriction enzymes
Connections made permanent by DNA ligase
How to ensure insertion?
Waitaminute!
What is problem?

18. Transforming Prokaryotes vs. Eukaryotes
Difficulty in inserting genes from eukaryotes into prokaryotes (and vice versa) in terms of
getting past plasma membrane = transformation
How to do this? What can use (from learned?)
Viruses (retrovirus), heating, electroporation
getting them to function…why?
Different regulation mechanisms
Prokaryotes = operons
Eukaryotes = enhancers, etc.
Often use expression vectors segments of DNA/RNA that will make sure gene is turned on after transformation and insertion

19. Let’s do an activity to showcase recombinant DNA!
Will come back to PPT in last 20 minutes of class

20. Genomic Libraries: Storing genes
Can store genes in a plasmid library or in BAC (Bacterial artificial chromosomes)
Can use reverse transcriptase to make cDNA (complementary)  cDNA library
Different because doesn’t have introns

21. Directly Identifying Genes (Probes)
With library, when look at a new organism’s genome, we can identify what genes it has using: nucleic acid hybridization
Construct a nucleic acid probe: short single-stranded DNA complement to gene of interest  tagged fluorescent

22. Nucleic Acid Probe Hybridization
When mix probe (that came from gene of interest) with a library, can identify gene!
Why not sequence?
More modern (problems with variation)

23. Polymerase Chain Reaction (PCR)
Amplifying DNA directly
Instead of putting gene of interest in bacteria, and having WHOLE cells reproduce to reproduce DNA…
Isolate DNA within a cell and make it
PCR can produce billions of copies of a target DNA segment in a few hours
Bacteria would take days to make the same amount (reproduce whole cell)

24. PCR Steps
1) Denaturation:
Heat DNA = (unzip)
2) Annealing: cool to allow (DNA) primers to attach (primers specific for target sequence)
3) Elongation: allow DNA polymerase to work (denature?)
Repeat Cycle (exponential)

25. Biotechnology at its best
PCR machines automate the cycles
Insert DNA with target gene
Insert primers
Machine runs automatically thru cycles
Was designed/engineered by Kary Mullis (won Nobel Prize)