Transcript and a regulatory gene

LABORATORY 2A: HOW DO YOU BEGIN
TO CLONE A GENE?
LSSI Alum,
Shawn Hurley
Resource Links/Additional Background
https://www.dnalc.org/view/15476-mechanism-of-recombination-3d-animation-with-with-basicnarration.html
http://highered.mheducation.com/sites/0072556781/student_view0/chapter14/animation_quiz_
2.html
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
Complete Genetic Engineering
Sequence
Lab 1: Tools of the Trade – Pipetting and Gel
Electrophoresis
Lab 2A: Restriction Digest (cut pARA-R)
Lab 4A: Confirmation (gel electrophoresis)
Lab 5A: Transformation (introduce pARA-R into
bacterial host cells)
Lab 6: Culture transformed bacteria, isolate and
purify the protein
Goals of Lab 2A
Logistical (students will coordinate procedural steps
necessary to):
• Perform restriction digest on recombinant plasmid
(pARA-R) in order to indirectly confirm its identity
and possession of the red fluorescent protein gene
Educational (students will be able to):
• Identify the common characteristics of plasmids
• Explain how plasmids are used as vectors in gene
cloning/expression
• Describe the function of restriction enzymes
• Explain how restriction enzymes are used to create
recombinant plasmids
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
http://www.pbslearningmedia.org/resource/biot11.sci.life.gen.genengdna/geneticengineering-and-working-with-dna/
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.
http://highered.mheducation.com/sites/dl/free/0072421975/196644/restriction_endonucl
eases.html
What are restriction enzymes?
Characteristics:
• Recognition sites are palindromic sequences, usually 4-8
nucleotides in length
5’ – G A A T T C – 3’
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)
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))
https://highered.mheducation.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/fr
ee/0072437316/120078/bio38.swf::Early%20Genetic%20Engineering%20Experiment
Lab 2A – Digesting pARA-R
(cutting the recombinant plasmid)
• Purpose: to produce DNA fragments of appropriate
size that will confirm the presence of the rfp gene.
– Will need to cut recombinant plasmid (construct)
• pARA-R – has the rfp gene with promoter sequence (pBAD), an
antibiotic resistance gene for ampicillin (ampr) and a regulatory
gene (araC) that codes for arabinose activator protein
- Arabinose is a sugar that induces transcription of the rfp
gene by working with arabinose activator protein
– Will use two restriction enzymes on the construct,
liberating the rfp insert from the remainder of the
plasmid/vector
• BamHI
• HindIII
Recombinant plasmid (pARA-R)
Lab 2A – Digesting pARA-R
Expected Resulting Digest Fragments
BamH
I
Hind III
4,495 bp
Hind III
807 bp
BamH I
Safety-Lab 2A
• Use laboratory coats, safety glasses and gloves as
appropriate
• Avoid restrictive clothing and open-toed shoes
• No eating or drinking in the lab
• Make sure that students are familiar with the
operating instructions and safety precautions
before they use any of the lab equipment
• Check all MSDS (Material Safety Data Sheets) for all
chemicals and reagents in the lab before preparing
and running the lab
www.amgenbiotechexperience.com
Lab & Aliquoting Guide-Lab 2A
Reagents/Supplies
1.4 ml 2.5X Buffer/class (2.5xB)
Aliquot
20ul/group
Storage Temp
4o
110 ul of recombinant plasmid
(RP)/ class (pARA-R)
65 ul of Restriction Enzyme/class
(RE)
10ul/group
-20o
5ul/group
-20o
12mL of DI water/ kit (dH20)
1ml/group
RT
Equipment/Supplies
10 Student boxes with the following:
1 p20 micropipette
1 p200 micropipette
1 p1000 micropipette
1 waste and
1 ice bucket
4 Mini centrifuges
1 Water bath
2 Floating racks
www.amgenbiotechexperience.com
1 microfuge rack
1 bag of microfuge tubes
1 bag of microfuge tubes
1 box of refillable tips (2 ul-200 ul)
Notes
Keep this for all Labs
Completing Lab 2A
Teacher Tips –
*make sure students label with
initials/group
*Remind them of mixing techniques
*Make sure centrifuge is balanced
before running
*Identify for students when to use a
new tip
Completing Lab 2A
(pg. C-15 in teacher manual)
Label tubes (“R+” & “R-”)
Add 4.0 uL 2.5xB into each
Add 4.0 uL of RP into
each tube
Add 2.0 uL RE into R+
tube and mix
Add 2.0 uL dH2O in R- tube
and mix
Centrifuge all tubes
Put all tubes in floating
rack and set in 37oC water
bath for 60 mins.*
Remove and place tubes
into freezer overnight
* - if time is limited, reducing the
digest time to 20-30 minutes is
aaceptable
Teacher Video Resources
• Mixing two solutions
video:
https://www.amgenbiotechexperience.
com/curriculum/curriculumresources/mixing-two-solutions
• Digestion video (different
digest, but good
techniques):
https://www.youtube.com/watch?v=Gs
Wo8dCivWs
• How restriction enzymes
work (good, short):
https://www.youtube.com/watch?v=lW
XryzgRces
• Longer, overall of lab 2
created for absent
students (screen cast):
https://www.youtube.com/watch?v=4
wbStjWEM8A
• Fun one from MIT. Covers
whole process 1st half
good for Labs 2&3:
•
https://www.youtube.com/watch?v=nf
C689ElUVk
***remember, sticky end issue here
Paper Plasmid Activity
Modeling Activity: Choose
the appropriate restriction
enzyme(s) to use to insert
the vasopressin gene into a
plasmid-based vector
Recombinant DNA Synthesis Handout (PDF) (PPT) – These may take
several minutes to download