Transcript Lab 6A - artesiascience

Bacterial Transformation
Lab 6A
2007-2008
Our Goals
1. Insert foreign gene into plasmid
2. Insert recombinant plasmid into
3.
bacteria
Grow transformed bacteria
Questions We Need Answered
1. Did the bacteria take up the plasmid?
2. If so, did the gene of interest get
inserted into the plasmid?
Analyzing our plates at the
end of the lab will answer
these questions and tell us if
we achieved our goals
Essential Knowledge
What must you know to
understand this lab?
Antibiotic Resistance
 Resistant bacteria can survive in the
presence of antibiotics
 We will be using ampicillin
 Our plasmid is resistant to ampicillin
Lac-Z gene
 Lactose = glucose + galactose
 Lactose is broken down by the Lac-Z

gene
Lac-Z will also digest similar
compounds
X gal
 X gal is a compound that is similar to
lactose
 X gal turns blue when it is digested by
the Lac-Z gene
Engineered Plasmids
 We will be using pBlue
Lac-Z
gene
Ampicillin Resistance
gene
Restriction Enzymes
 Our plasmid DNA and our foreign DNA
arrive pre-cut by the same restriction
enzyme
 The restriction enzyme made a single
cut in the plasmid
 The restriction site is within the lac-Z
gene
Restriction
site
Lac-Z
gene
Ampicillin Resistance
gene
We will be using 3 types of agar
1. LB
2. LB + amp
3. LB + amp + X gal
Each Group Will Run Six Plates
LB
LB
- plasmid
+ plasmid
LB/amp
LB/amp
- plasmid
+ plasmid
LB/am/X-gal
LB/am/X-gal
- plasmid
+ plasmid
How will we know if we are successful?
LB Plates
 There is nothing in the LB agar to
interfere with growth.
 Growth on both the + and – plates
indicates the bacteria are alive and well.
 A “lawn” is too many colonies to count
- plasmid
+ plasmid
 What if nothing grows on either LB
plate?
LB/amp Plates
 Only bacteria that have taken up the
plasmid will grow on the plates with
ampicillin
 How do you know?
If the plasmid is inserted into the bacteria,
 it will become antibiotic resistant and
will grow on the ampicillin plate
only transformed
bacteria grow
LB + amp
LB/amp Plates
 No growth on – confirms ampicillin is
working because bacteria without the
plasmid are not resistant and die
 Bacteria growing on + contain the
plasmid and are amp resistant
- plasmid
+ plasmid
LB/amp Plates
 Would you expect isolated colonies or
a lawn on the + ? Why?
LB/amp Plates
 What if your – shows unexpected
growth?
 Can you tell by looking at + which ones
are recombinant?
LB/amp/X-gal
 Color of colonies on X-gal plate will tell
you if gene of interest has been
inserted into the plasmid or not
 Blue colonies indicate Lac Z gene is
digesting X-gal
 White colonies indicate Lac Z gene in
not functioning
LB/amp/X-gal
 If ends of lac Z gene come back
together and are resealed by DNA
ligase during the transformation
process, the Lac Z gene will be
functional and will turn X gal blue.
LB/amp/X-gal
 If the gene of interest is successfully
inserted between the free ends of the
plasmid, the Lac Z gene will be
“broken”.
 If the Lac Z gene is interrupted, X-gal
will not be digested and the colonies
will remain white
If the foreign DNA is inserted into the plasmid,
 The Lac-Z gene will be interrupted and
bacteria will stay white
inserted
gene
of interest
“broken”
LacZ gene
recombinant
plasmid
amp
resistance
LB + amp + X gal
Blue vs. White on X-gal Plate
Bacteria take up plasmid
Functional LacZ gene
X gal is being digested
Bacteria make blue color
Bacteria take up recombinant plasmid
Non-functional LacZ gene
X gal is NOT being digested
Bacteria stay white color
 What if you just see blue colonies on + ?
 Should you ever see blue colonies on - ?
 Should you see isolated colonies or a
lawn on the X gal plate? Why?
- plasmid
+ plasmid
What do we really want to see?
White Colonies on X-gal !
LB + amp + X gal
SUCCESS!
Any
Questions?