1. MAKING A RECOMBINANT PLASMID Transformation of Bacteria using plasmids & restriction enzymes By Kelly Riedell Brookings Biology
Blue edged slides from slide show by Kim Foglia
Recombinant Plasmid activity modified from

2. Big Idea 3: Living systems store, retrieve, transmit and respond to information essential to life processes. Enduring understanding 3.A: Heritable information provides for continuity of life. Essential knowledge 3.A.1: DNA, and in some cases RNA, is the primary source of heritable information e. Genetic engineering techniques can manipulate the heritable information of DNA and, in special cases, RNA. To foster student understanding of this concept, instructors can choose an illustrative example such as: • Electrophoresis • Plasmid-based transformation • Restriction enzyme analysis of DNA • Polymerase Chain Reaction (PCR) f. Illustrative examples of products of genetic engineering include: • Genetically modified foods • Transgenic animals • Cloned animals • Pharmaceuticals, such as human insulin or factor

3. BACTERIA HAVE PLASMIDS
Image from:
BACTERIA HAVE PLASMIDS
Small, extra-chromosomal, self-replicating, rings of double stranded DNA that can carry genes for conjugation and antibiotic resistance Can be used in genetic engineering to transfer genes from other organisms into bacteria

4. GENETICALLY MODIFIED ORGANISMS
SLIDE MODIFIED FROM KIM FOGLIA biology.com
GENETICALLY MODIFIED ORGANISMS
“Golden” rice (Vitamin A) Bt-corn-resists insect pests Frost resistant bacteria
Toxic cleanup bacteria

5. Genetically modified organisms (GMO)
SLIDE MODIFIED FROM KIM FOGLIA biology.com
Genetically modified organisms (GMO)
enabling plants to produce new proteins
Protect crops from insects: BT corn
corn produces a bacterial toxin that kills corn borer (caterpillar pest of corn)
Extend growing season: fishberries
strawberries with an anti-freezing gene from flounder
Improve quality of food: golden rice
rice producing vitamin A improves nutritional value
For example, a transgenic rice plant has been developed that produces yellow grains containing beta-carotene.
Humans use beta-carotene to make vitamin A.
Currently, 70% of children under the age of 5 in Southeast Asia are deficient in vitamin A, leading to vision impairment and increased disease rates.

6. How can plasmids help us?
SLIDE FROM KIM FOGLIA biology.com
How can plasmids help us?
A way to get genes into bacteria easily
insert new gene into plasmid
insert plasmid into bacteria = vector
bacteria now expresses new gene
bacteria make new protein
transformed bacteria
gene from other organism
recombinant plasmid
vector
plasmid
cut DNA +
glue DNA

7.  How do we cut DNA? Restriction enzymes restriction endonucleases
SLIDE FROM KIM FOGLIA biology.com
How do we cut DNA?
Restriction enzymes
restriction endonucleases
discovered in 1960s
evolved in bacteria to cut up foreign DNA
“restrict” the action of the attacking organism
protection against viruses & other bacteria
bacteria protect their own DNA by methylation & by not using the base sequences recognized by the enzymes in their own DNA 

8. RESTRICTION ENDONUCLEASES
ENZYMES cut DNA at a specific “palindrome” sequences
= restriction site
Produce sticky ends
Different kinds of DNA cut with same enzyme will
have the same “sticky ends” and can be joined
Different enzymes recognize & cuts different sequences
100’s of different enzymes named after organism they are found in
EcoRI, HindIII, BamHI, SmaI

10. Plasmid Image from: http://faculty. ccbcmd
PLASMID DNA
Cut the PLASMID DNA strips apart and tape them together in one long strip.
Use the numbers to help you match ends in the correct sequence.
Letters should all be facing in the same direction.
Tape the two ends of the long strip together to form a circular plasmid with the letters FACING OUT
Dark area represents ORIGIN OF REPLICATION

11. HUMAN INSULIN DNA
Cut the HUMAN INSULIN DNA strips apart and tape them together in one long strip.
Use the numbers to help you match ends in the correct sequence.
Letters should all be facing in the same direction.
Remember: Human chromosomes are LINEAR!
Dark area represents INSULIN GENE

13. Compare the sequences of base pairs on each of the RESTRICTION ENZYME SEQUENCES provided to the DNA sequences on the PLASMID DNA.
If you find the same sequence, MARK THE LOCATION and the CUT PATTERN
on BOTH THE ENZYME and the
PLASMID DNA using the same color.
Repeat comparison using RESTRICTION ENZYME SEQUENCES and HUMAN DNA sequence.

14. OBJECTIVE FIND a RESTRICTION ENZYME SEQUENCE that will:
Cut the PLASMID DNA in ONLY ONE LOCATION
NOT cut the ORIGIN OF REPLICATION SEQUENCE in the PLASMID DNA

15. IF CUT PLASMID IN MORE THAN ONE PLACE

16. IF CUT PLASMID IN MORE THAN ONE PLACE

17. IF CUT PLASMID IN MORE THAN ONE PLACE
If plasmid has
ORIGIN OF REPLICATION plasmid can copy itself when cell divides
Can grow a whole vat of bacteria producing human insulin.
Bacteria that have plasmids
w/o ORIGIN OF REPLICATION can divide, but plasmid can’t copy itself.
As cells divide, get more and more cells w/o human insulin gene.

18. OBJECTIVE ALSO FIND a RESTRICTION ENZYME SEQUENCE that will:
NOT cut the INSULIN gene
Make one cut in the HUMAN DNA on either side of AND as close as possible to the INSULIN GENE sequence

19. CREATE YOUR PLASMID
After you have examined ALL of the enzyme sequences
Provided, decide the best one to use.
Make cuts on both the PLASMID and HUMAN DNA to create “sticky ends” as shown on the ENZYME sequence.
USE LIGASE to join the “sticky ends” of the PLASMID
and INSULIN gene together.
CONGRATULATIONS! You have created a recombinant plasmid which can now be inserted into a bacterial cell to produce human insulin!

21. Grow bacteria…make more
SLIDE FROM KIM FOGLIA biology.com
Grow bacteria…make more
transformed bacteria
gene from other organism +
recombinant plasmid
vector
plasmid
grow bacteria
harvest (purify) protein

22. PROBLEM !!!!!!!
EUKARYOTIC mRNA is edited before it is translated into a protein
NO POST TRANSCRIPTIONAL PROCESSING IN PROKARYOTES NO INTRONS NO EDITING ENZYMES
CAN’T USE EUKARYOTIC DNA DIRECTLY
NEED EDITED mRNA to make human proteins

23. reverse transcriptase
“TRICKY” SOLUTION
Don’t start with human DNA…
Use mRNA for insulin to make cDNA(complementary DNA)
copy of the gene without the introns
How do you go from RNA  DNA?
reverse transcriptase from RNA viruses
retroviruses
cDNA
reverse transcriptase
IMAGES FROM KIM FOGLIA

24. REVERSE TRANSCRIPTASE

25. Plasmid- 1 cut NO cuts on human DNA
Plasmid- 1 cut 3 cuts on human DNA
Plasmid- 1 cut 2 close cuts on human DNA
Plasmid- 2 cuts 2 cuts on human DNA
Plasmid- 1 cut CUTS INSULIN gene