Download MCB_151_Exercise 10_Glow

Producing a Strain of
E.coli that Glows in the
Dark
Exercise 10
Announcements
 Post
Lab 10 and Pre Lab 11 is due by your next lab
 LNA Glow
Lab is assigned today, and is due next week
in lab
 Exam
II is during your lab period the week of November
30 and will cover Exercises 8-11
 The
Final Exam is scheduled for Friday, December 11
from 8:00-11:00 AM. Locations TBA. If this conflicts with
any of your other final exams, you must fill out the Final
Exam Conflict Request Form by 5 PM on 11/9/15.
Goals
 To
create an ampicillin-resistant population
of E. coli by introducing a plasmid that
contains an ampicillin resistance gene
 Understand the lux Operon and how it is
used to create luminescent bacteria
 Understand the pUC18 plasmid and how it
serves as a control in this experiment
Penicillin
 One
of the most important anti-infective
agents used in clinical medicine
 Interferes with bacterial cell wall synthesis
 Over 500 semi-synthetic penicillins have been
made during the past 30 years
Plasmids
 Plasmids
are small circular DNA
molecules that exist apart from the
chromosomes in most bacterial species
 Many contain genes that enable
bacteria to survive and prosper in
certain environments b/c they carry
genes that provide resistance to
antibiotics
pUC18 Plasmid
 Circular
DNA that contains 2686 nucleotides
(very small)
 Contains an ampicillin-resistance gene
pUC18 Plasmid
Transformation
Process of introducing plasmids into living
bacterial cells.
Procedure:
•Treat bacteria
with calcium
chloride
•Add the plasmid
DNA
•Select for the
transformed
bacteria on
antibiotic media
lux Operon
 Contains
two genes that code for luciferase and
several genes that code for enzymes which produce
luciferins
R
T
C
D A B
E
G
Rib
The lux Operon. The lux operon contains two genes for the luciferase enzyme (A and
B). This enzyme is composed of two different polypeptide chains. The operon also
contains several other genes (R, T, C, D, E, G, And Rib) that are thought to code for
enzymes which produce the substrates for the light-emitting reaction. These substrates
are called luciferins and are long chain fatty aldehydes.
The Exercise
 Part

I: Preparation of Competent Cells
Review procedure on page 122 of your lab manual. This has
been done for you prior to starting the experiment.
 Part
II: Uptake of Plasmid by competent cells
 Part III: Selection on ampicillin plates

Follow procedure in your lab manual. Note that to store your
plates in the dark, you will put them in a paper sack and then
in a box designated for your section
Protocol
Your cells must stay on ice at all times !!!!
•
Transfer 50 μL from the EC tube to each of the tubes labeled C
(control), L (lux), and NP (no plasmid)
•
Incubate tubes for 20 min on ice
•
Incubate tubes for 30 sec at 42°C
•
Incubate on ice for 5 min
•
Add 500 μL of Nutrient Broth to each tube and incubate at
37°C for 30 min at 230 rpm
•
Spread 100 μL of each tube into an agar plate containing
ampicillin (one plate per tube) (label plates)
•
Invert plates and incubate in dark
Results
Part IV:
Examination of cultures in the dark
 You
will have to attend open hours on:
Section
Open Hour Time (210 Noyes)
A
Thursday (11/12); 9 AM – 3 PM
B
Thursday (11/12); 11 AM – 5 PM
C
Thursday (11/12); 2 PM – 8 PM
D
Thursday (11/12); 2 PM – 8 PM
E
Thursday (11/12); 5 PM – 8 PM
Friday (11/13); 9 AM – 11 AM
F
Friday (11/13); 11 AM – 3 PM
G
Friday (11/13); 11 AM – 3 PM
H
Friday (11/13); 11 AM – 3 PM