Transformation
AP Big Idea #3: Genes and
Information Transfer
connected with AP Big Idea
#1 (Evolution) & #2 (Cellular
Processes)
How has our understanding
changed?
What distinguishes stem cells
from differentiated cells?
What types of genes are turned
on/off as the path towards
differentiation occurs?
Its all about regulation!

Gene Regulation in Bacteria



Operons
Use of bacterial gene regulation systems in
biotechnology
Gene Regulation in Eukaryotes: (We’ll come
back to this in a later powerpoint!!!)



Regulation at the DNA Level
Regulation at the transcription level
Regulation at the translation level
Bacterial Operons
 Operon:
set of genes that control the
production of a certain protein product
needed by the cell under specific
conditions.


Inducible
Repressible
 What
do you think is the difference
between these?
Inducible: Example LAC
How does
the
presence of
lactose
“induce”
transcription
?
Repressible: TRP operon
Why are operons necessary?
 What
is the advantage of E. coli having
evolved?


A lac operon?
A trp operon?
Using Bacterial Genetics for
Biotechnology
 Transformation:

Bacteria can uptake eukaryotic DNA and
become gene factories.
What is a plasmid?
Basic Structure of a Plasmid
What does a plasmid do in a
bacteria? “Sex”
Selectable markers
 Resistance
to antibiotics (like kanomycin)
 If bacteria gets plasmid; it will grow in
presence of antibiotic.
 Screens out bacteria that don’t receive
plasmid.
Origin of Replication
 Bacteria
divide
rapidly to form
colonies.
 They must be
able to
replicate the
plasmid into
their daughter
cells.
Making a plasmid:
 Restriction


enzymes:
Enzyme present in bacteria that cuts DNA
at certain points (used to protect bacteria
from viruses)
Used in biotechnology to cut open a
plasmid and glue in a gene of interest
 Ligase:

Enzyme used to glue in inserted piece of
DNA (where did we talk about this enzyme
before?)
Problems that can be
encountered
The hard part is done!
 We
bought pre-engineered pGLO
plasmid.
 Plasmid was engineered and then copied
by BioRAD!
Engineering
a bacteria
Steps we will do:
 Heat
shock bacteria to add plasmid.
 Grow (clone) bacteria to give them a
chance to replicate plasmid (E. coli
populations double every 20 minutes)
 Incubate bacteria and allow them to
produce protein=green fluorescence
protein
 Use an operon to turn on/off the presence
of protein production!
pGLO Lab:
 How
can we alter the transformation
process to improve transformation
efficiency?

Each lab group
What you must know before
the lab:

Research the pGLO plasmid. Be able to identify:



The operon, the selectable marker, the inserted
gene.
Read through the manual (available on BioRad’s
website and through a quick web search…pGLO
student manual)
Predict what will happen on each plate you
make:




LB
LB/Amp
LB/Amp
LB/Amp/Ara
-pGLO
-pGLO
+pGLO
+pGLO
Lab Day:
 Decide
how will your group “tweak” the
procedure to attempt to increase
transformation efficiency.
 Identify the expected result for each
plate and understand why each control is
used.
 Identify in your lab notebook how you will
calculate transformation efficiency (for
your plate that produces GFP) the next
day.
Data and Analysis Day:
 Calculate
control transformation
efficiency. Put in spreadsheet (we will
have 3 sets of data for class control)
 Calculate the average transformation
efficiency for your 3 experimental titles.
 In your groups, make sure you understand
the specific purpose of your group’s lab!
Follow-up:



This lab is a model of using transformation to
produce a protein product.
Find a real life example of how bacterial
transformation was used (or how GFP was
used) and explain understanding this model is
useful (implications and connections!)
Remember…we are using Semester 2 Lab
rubric…Here is a chance to show the skills you
learned as a big lab group! 