Name(s):__________________________
Paper Plasmid Lab
In the exercise you will use paper to simulate the cloning of a gene from one organism into a bacterial plasmid using a restriction enzyme . The plasmid (puc18 plasmid) can then be used to transform bacteria so that it now expresses the new gene and produces a new protein.
1. From the WHITE paper, cut out the bacterial plasmid DNA in a long strip.
2. Using tape, attach the ends together to make a loop to simulate the curricular DNA of a plasmid.
3. From the GREEN paper, cut out the Jellyfish Glo gene (GFP - green florescent protein) in a long strip. Leave it as a straight strip (This gene is not from a bacterium, so it is not circular). The start and stop sequences for transcribing the GFP gene are highlighted. These are needed to transcribe and then translate the gene properly. In addition, both the HindIII and EcoR1 restriction enzyme cutting sites are marked in bold on the Jellyfish GFP DNA. The two restriction enzymes and their respective restriction sites are listed below. These enzymes act as “molecular scissors” which cut the DNA at these sequences in the DNA.
Restriction
Enzyme
HindIII
EcoRI
Recognition site
(5’-3’)
A
↓
AGCTT
TTGCA
↑
A
G
↓
AATTC
CTTAA
↑
G
The six letter sequence represent the nitrogen base sequence that the enzyme
!
recognizes, and the ↓ or ↑ represents the place where the DNA will be cut by the
!
enzyme. For example, HindIII cuts between the first A and the second A whenever it
!
encounters the six base sequence AAGCTT.

4. Cut the GREEN jellyfish DNA as if you has used the restriction enzyme HindIII . Be sure to leave ‘sticky ends’.
5. Also, cut the WHITE puc18 plasmid DNA as if you have performed a restriction enzyme digest with HindIII.
Be sure to leave ‘sticky ends’.
6. Now you will incorporate the green GFP gene into the plasmid. Attach the sticky ends of the jellyfish GFP gene to the sticky ends of the puc18 plasmid and seal with the enzyme ligase (for our lab purposes.... scotch tape).
7. You have now successfully cloned a gene! You have a single bacterial plasmid with a new gene which can then be used transform a bacteria. The bacterium will now make the jellyfish GFP protein and glow under black light. Because bacteria reproduce by binary fission as often as every 20 minutes, in 24 hours 1 transformed bacteria can become over 4,722,366,482,869,465,213,696 glowing bacteria! (4 sextillion, 722 quintillion, 366 quadrillion, 482 trillion, 869 billion, 465 million, 213 thousand, 696.)

QUESTIONS:
1. What is a plasmid?
2. What are restriction enzymes? Where are they found naturally and what purpose do they serve?
3. How have we found a way to use restriction enzymes for scientific advancements in
DNA and biotechnology?
4. Why did we cut both DNA segments with the same restriction enzyme?
5. Why did we make sure the to include the start and stop DNA sequences for the GFP gene in our cut fragment?
6. What would have happened if we had cut both the plasmid and the GFP DNA with
EcoRI instead of HindIII? Be sure to look on the paper DNA sequences to find the
EcoR1 restriction enzyme cut sites.