biotechnology lab program
Labs 2, 3, 4, 5
Restriction Analysis of
pARA and pKAN-R
RFP Expression Sequence
Tara Bennett
Alia Qatarneh
Harvard University LS Outreach Program
timeline
•  8:30-9:45am
–  Intro to laboratories
–  Start Lab 2: 60 minute
incubation at 37°C
•  9:45-9:55am
–  Break
•  9:55-11:10am
–  Start Lab 3: 30 minute
incubation at 70°C
•  11:10-11:45am
–  Lunch
–  Ligation incubation during lunch
at 37°C
V.1.2.1
•  11:45-1:00pm
–  Start Lab 4
–  Run E-gels  observe results
•  1:00-1:05pm
–  Break
•  1:05-2:20pm
–  Start Lab 5: plating
–  Need H2O bath at 37°C
•  2:20-2:30pm
–  Wrap up
lab 2: restriction analysis of pKAN-R and pARA
•  What is a plasmid?
–  pKAN-R plasmid
–  pARA plasmid
•  What is a restriction enzyme?
–  BamHI restriction enzyme
–  HindIII restriction enzyme
•  What’s so special about these plasmids and enzymes?
–  Plasmids have antibiotic resistant genes!
•  So what?
–  BamHI and HindIII allow us to cut our plasmids in specific places
•  Again, so what?
V.1.2.1
lab 2: restriction analysis of pKAN-R and pARA
pKAN-R
5408 bp
rfp
Bam
H
I
702 bp
pARA
4058 bp
40 bp
lab 2: restriction analysis of pKAN-R and pARA
Restriction fragments after digest with Hind III and BamH I
BamH I
Hind III
4018 bp
BamH I
Hind III
4706 bp
BamH I
Hind III
702bp
Hind III
BamH I
40 bp
lab 2: pipetting chart
Tube
2.5x
Buffer
dH2O
pARA
pKAN-R
Enzyme
Mix
Total
Volume
A+
4uL
-
4uL
-
2uL
10uL
-
-
AK+
K-
4uL
4uL
4uL
2uL
4uL
-
-
2uL
-
4uL
4uL
TIPS:
• Make sure all tubes are correctly labeled
• Put group initials are somewhere on the tube
• Close tightly before putting them in the H2O bath
V.1.2.1
2uL
-
10uL
10uL
10uL
lab 2: restriction analysis of pKAN-R and pARA
Prediction for restriction gel
M K+ K- A+ A-
M K+ K- A+ A-
10000
8000
5000
4000
3000
2000
1500
1000
Rfp gene 500
lab 3: ligation of pKAN-R/pARA restriction fragments
BamH I
sticky end
3’
5’
3’
5’
sticky end
5’
5’
3’
3’
Hind III
BamH I
sticky end
Hind III
sticky end
3’
5’
3’
5’
lab 3: ligation of pKAN-R/pARA restriction fragments
Recombinant plasmid of interest
pARA-R
4720 bp
rfp
702bp
Lab 3: pipetting chart
Tube
A+
K+
5x
Ligation
buffer
Lig
4uL
4uL
3uL
dH2O
T4 Ligase
Total
Volume
2uL
2uL
15uL
TIPS:
• Do not throw out any tubes
• Make sure all tubes are correctly labeled
• Put group initials are somewhere on the tube
• Close tightly before putting them in the H2O bath
V.1.2.1
lab 4: confirmation of restriction digest
•  Time to run the gel!
–  Did the restriction enzymes “digest” the pKAN-R and pARA plasmids?
How can you tell?
–  Which lane has the ligation sample? How do you know?
–  Why is it important to have the A- and K- negative controls on the gel?
–  What is gel electrophoresis?
•  How are bands separated?
V.1.2.1
lab 4: confirmation of restriction digest
Confirmation of restriction and ligation
M K+ K- A+ A- L
M K+ K- A+ A- L
Lig 10000
8000
5000
4000
3000
2000
1500
1000
Rfp gene 500
40bp lab 5: transforming E. coli with recombinant plasmid
•  What are we trying to do in Lab 5?
•  What is a transformation
–  DNA manipulation and incorporation?
•  What are we trying to express?
•  How do we “express” this?
–  Sugar?
•  How the heck do we know it’s expressing?
V.1.2.1
transforming Escherichia coli with pARA-R
Competent Cells
pARA-R
Recombinant Plasmids
transforming Escherichia coli with pARA-R
Lipid bilayer
(inner)
Peptidoglycan
layer
Adhesion zone
Lipid bilayer
(outer)
Calcium ions
pARA-R
lab 5: preparing competent cells for transformation
Samples
Tube
Competent Cells
Plasmid: Lig
P+
50uL
10uL
P-
50uL
NONE
•  To the P+ tube:
•  Add 50 uL of comp cells and 10 uL of pARA-R plasmid
•  To the P- tube:
• Add 50uL of comp cells BUT NO PLASMID FROM LIG TUBE
•  Incubate on ice: 15 min
•  Heat shock: 42 degrees C for 45 seconds
•  Back on ice: 1 min
•  Add 150uL of LB broth to each tube and mix
•  Let them sit at RT for a bit works too if you only have 1 water bath and can't
switch the temp quickly.
•  PLATE!
•  Draw line down LB and LB/amp plates
•  Mark one side for P+ and the other for P•  LB/amp/ARA plate is only P+ sample
lab 5: preparing competent cells for transformation
Plating
•  Note the plate markings
•  LB only: 1 blue line
•  LB/ampicillin: 2 black lines
•  LB/ampicillin/Arabinose: 3 lines total (2 black, 1 red)
•  Label the bottom of the plate near the edge
•  Open the plates like clam shells
•  Remember: sample media should SKATE on the agar (don’t dig into it)!
http://www.teachersdomain.org/asset/biot11_vid_plating/
lab 5: preparing competent cells for transformation
STRESS sterile techniques!
•  Always follow the protocol carefully – know what
you’re doing
•  Work quickly – less time = less opportunities for
contamination
•  Do not leave any container (tube, plate) open any
longer than needed
•  Watch what your equipment touches –
•  All tips, tubes and spreaders in the “contaminated
waste” container at the end of the lab.
growth of transformed bacteria on various plates
P+ plates
LB
LB/amp
P- plates
No growth
LB
LB/amp
LB/amp/ara
Without arabinose:
AraC protein prevents rfp transcription by causing loop to form in promoter region: mRNA
transcription cannot occur  rfp OFF!
With arabinose:
Arabinose-AraC protein complex (AAPC) prevents looping
AAPC helps align RNA polyermase to promoter site where regulation of rfp expression occurs
Transcription HAPPENS, makes mRNA, translated into mFP  rfp ON!
RFP expression
araC gene
PBAD
Transcription
mRNA
Translation
araC protein
rfp gene
RFP expression
araC protein prevents RFP transcription by causing
a loop to form in the region of the r fp gene
araC gene
araC protein
PBAD
rfp gene
RFP expression
arabinose
Arabinose – araC protein complex prevents DNA looping
and helps to align RNA polymerase
on the promoter site (PBAD).
Translation
RNA polymerase
arabinose
araC protein
araC–protein
complex
mRNA
Transcription
araC gene
PBAD
rfp gene