Agenda
• Learn why biotechnology is important in modern-day
medicine, agriculture, and environmental
remediation
• Learn about about plasmids & restriction enzymes as
tools in molecular biology/biotechnology/genetic
engineering
• Learn about precise measurement of ultra-small
volumes using a micropipette
• Watch and become familiar with the procedures for
plasmid isolation and restriction enzyme digest
Important Vocabulary/Terms
-Plasmid
-Vector
-Insert
-Construct
-Digest
-Ligase
-Palindromic
-Restriction Enzyme
-Sticky Ends
-Complementary Bases
-Origin of replication
-Promoter
-Antibiotic resistance gene
-Regulatory gene
What is a plasmid?
• Comparatively small circular
double-stranded DNA
molecules of bacterial origin
• Range in size from 1,000 to
200,000 base pairs (bp)
• Independent of bacterial
chromosome, carrying
“nonessential” genes
Plasmid Features:
• Origin of replication
• Promoter
• Antibiotic resistance
• Multiple cloning site
(polylinker)
• Regulatory element(s)
Function:
- Region responsible for
initiating the copying of
plasmid DNA
- Site to which RNA polymerase
binds to begin transcription
- Gene coding for a product
that confers antibiotic
resistance
- Unique restriction sites allow
for the digestion of plasmid &
introduction of insert (foreign
gene)
- Gene coding for a product
that regulates transcription of
the insert
promoter
regulatory
gene
MCS
(with insert)
antibiotic
resistance
gene
origin of replication (Ori)
Plasmids as Vectors
What are restriction enzymes?
• Catalytic proteins that function like molecular scissors,
cutting double-stranded DNA at distinct recognition sites
that are usually unique to a particular enzyme.
What are restriction enzymes?
Characteristics:
• Recognition sites are palindromic sequences, usually 4-8
nucleotides in length
Cut sites
5’ – G A A T T C – 3’
are NOT
aligned
3’ – C T T A A G – 5’
• Cleave covalent bonds of sugar-phosphate backbone
• If enzyme is a staggered cutter, generates sticky ends
(unpaired overhangs capable of hydrogen bonding with
complementary bases) 5’ – G
A A T T C – 3’
3’ – C T T A A
G – 5’
• Nonemclature based on source bacterial species & strain
1st letter
roman numeral designates
E
co
R
I
of genus
order of discovery
1st two
strain
(Escherichia)
letters of species (coli)
What about aligned cuts?
Characteristics:
• Recognition sites are still palindromic sequences, usually 48 nucleotides in length
Cut sites
5’ – C C C G G G – 3’
ARE aligned
3’ – G G G C C C – 5’
• Covalent bonds of sugar-phosphate backbone are cleaved
as before
• In the above example, the enzyme is now a blunt cutter,
generates blunt ends (NO overhangs – any two blunts ends
can match up…not based on complementary/matching
sequence)
5’ – C C C
G G G – 3’
3’ – G G G
C C C – 5’
Application of These Molecular Tools
– Scientists can build designer plasmids that contain specific restriction
sites
– This allows scientist to cut out and recombine genes to allow for
cloning and gene expression (requires cutting each DNA sample with
same restriction enzyme(s))
Gene Cutting & Splicing Simulator
• Visit the following simulation activity site:
• http://glencoe.mheducation.com/sites/dl/free/007880284
9/383937/BL_22.html
• Perform two simulations by following the instructions
provided by the simulator:
1. Splice the human insulin gene into E. coli bacteria
2. Splice the firefly luminescence (“glow-in-the-dark”) gene
into tobacco plant
Tip: Make sure you select an enzyme (A-J; descriptions of
where each will cut are provided by the simulator) that will
cut outside of the highlighted gene sequence
Our Recombinant Plasmid (rpARA)
Regulatory gene – codes
for repressor protein
that controls the expression
of rfp/tomato (insert) gene
Promoter for
insert gene
Restriction
Enzymes
Ampicillin resistance gene –
codes for b-lactamase enzyme that
destroys the antibiotic called ampicillin
Insert rfp gene – codes
for Red Fluorescent Protein
Why “Tomato”?
• The name “tomato” is a nickname derived from
the shared color of tomatoes and the gene’s
protein product, Red Fluorescent Protein (RFP)
• The tomato gene (a.k.a. rfp gene) is NOT derived
from tomato plants; it originates from a species
of sea anemone that glows/fluoresces red in the
ocean upon exposure to UV light from the Sun.
• With this gene as our insert in the recombinant
plasmid, rpARA, we can give it to any living cell
and cause that cell to make red fluorescent
protein and glow/fluoresce red upon expression
of the gene
Extracting & Digesting rpARA
(isolating and cutting the recombinant plasmid)
• Purpose: to collect plasmid DNA from bacteria,
perform a restriction enzyme digest, and produce
DNA fragments of appropriate size that will confirm
the presence of the rfp/tomato gene.
– Will need to perform a plasmid isolation protocol
• Miniprep using alkaline lysis
– Will use two restriction enzymes on the isolated plasmid,
liberating the rfp/tomato insert from the remainder of the
plasmid (a.k.a. vector):
• BamH I
• Hind III
Plasmid Isolation from E. coli
• First, read the introduction on pg. 89 of your lab manual and
then watch the tutorial on how to use the various
micropipettes:https://www.youtube.com/watch?v=uEy_NGDfo_
8
• Complete the virtual micropipette activity & take the quiz at the
end as practice:
http://www.virtual-labs.leeds.ac.uk/pres/micropipettes/
• Next, view the procedure, outlined on pages 94-96, executed in
a video format (follow along as you watch) – Note: steps 11-18
in the actual lab protocol require a DNA precipitation and
washes…this has been replaced in the video with a spin column
purification (video time index = 7:25) :
https://www.youtube.com/watch?v=8xEDEJ0DHFA&t=181s
Plasmid Double Digestion
• Lastly, read the introduction on pg. 97 and watch a similar
procedure to that on pgs. 98-99 demonstrated in the
following video – Note: the actual lab protocol is for a
“double digest” of bacterial plasmid DNA (rpARA) where
the enzymes, BamH I & Hind III are combined in the same
reaction at the same time rather than
individually/separately:
https://www.youtube.com/watch?v=GsWo8dCivWs&t=128s
How will we know if we were successful in
our digestion of the plasmid?
1. We must confirm the double digestion of the
rpARA plasmid produced fragments of expected
size.
2. We must confirm that the negative control did
not cut the rpARA plasmid.
We will confirm these two points next week
with a methodology know as agarose gel
electrophoresis; Please answer the questions on
page 100 of your lab manual.
www.amgenbiotechexperience.com