Bacterial
Transformation
Lab
Restriction enzymes
Restriction enzymes are isolated from bacteria.
Bacteria use them to cut up Phages.
Each enzyme is specific for a different phage and has a unique cut site.
They often leave Sticky ends
This makes them a great tool in recombinant work.
Where do I get them?
Restriction enzymes are
available in a wide
variety of
cut sites.
How Do You Know Which Ones To Use?
These restriction maps are for 2 different plasmids.
Using Vectors - for our own
means of transfer!
Transformation
Once a plasmid has been modified, a bacteria can be “transformed” by the uptake of the plasmid.
In a lab setting, we can facilitate this using a technique called “heat shock”.
http://www.dnalc.org/resources/animations/tran sformation2.html
More
application?
Plasmid called pGlow
When a gene is
activated it
glows.
Our lab
Plasmid pAMP
Is resistant to an anti- biotic
We could ligate a
gene into it with
restriction enzymes…
but we did
n ’t!
Using these
techniques, we can insert and clone human or other eukaryotic DNA.
But, how do you keep track of the desired DNA or gene?
•
Only bacteria w/
plasmid grow
•
Only bacteria w/ a inserted gene
(disrupted lacZ) turn white
Isolate plasmid DNA and human DNA.
Cut both DNA samples with the same restriction enzyme.
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids and many nonrecombinant plasmids.
Bacterial cell lacZ gene (lactose breakdown)
Human cell
Restriction site ampR gene
(ampicillin resistance)
Bacterial
plasmid Gene of
interest Sticky
ends Human DNA
fragments
Recombinant DNA plasmids Introduce the DNA into bacterial cells
that have a mutation in their own lacZ gene.
Recombinant bacteria Plate the bacteria on agar
containing ampicillin and X-gal.
Incubate until colonies grow.
Colony carrying non- recombinant plasmid with intact lacZ gene
Colony carrying recombinant plasmid with disrupted lacZ gene
Bacterial clone Isolate plasmid DNA
and human DNA.
Cut both DNA samples with the same restriction enzyme.
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids and many nonrecombinant plasmids.
Bacterial cell lacZ gene (lactose breakdown)
Human cell
Restriction site ampR gene
(ampicillin resistance)
Bacterial
plasmid Gene of
interest
Sticky ends
Human DNA fragments
Recombinant DNA plasmids Introduce the DNA into bacterial cells
that have a mutation in their own lacZ gene.
Recombinant bacteria
Plate the bacteria on agar containing ampicillin and X-gal.
Incubate until colonies grow.
Colony carrying non- recombinant plasmid with intact lacZ gene
Colony carrying recombinant plasmid with disrupted lacZ gene Bacterial
clone
The Steps of Cloning a Gene
1. Isolate the GENE that will be cloned.
2. Cut the GENE and a VECTOR (viral or plasmid DNA) with the same RESTRICTION ENZYME.
3. LIGATE the GENE into the VECTOR (plasmid).
4. TRANSFORM a competent BACTERIA (or yeast)
5. Grow the BACTERIA on a SELECTIVE growth media that identifies bacteria with the
plasmid.
Cloning DNA in a test tube = PCR
In order to sequence
DNA, you must replicate DNA outside of a cell.
Polymerase Chain
Reaction, or PCR does this using a
thermocycler, and heat stable polymerase,
(taq), isolated from thermophilic bacteria.
http://www.dnalc.org/resources/animations/pcr.ht ml
http://www.sumanasinc.com/webcontent/animations/co ntent/pcr.html
http://www.dnalc.org/resources/3d/19-polymerase-chain- reaction.html
https://www.youtube.com/watch?v=Bkv8c978DWU
https://www.youtube.com/watch?v=FpxwJNNufko
The Steps in Our Lab?
1. Transformation of E. coli with pGreen.
2. Select resistant colonies, and grow over night.
3. Isolate the cloned plasmid from the bacteria by doing a miniprep.
4. Isolate the GFP protein.
5. Cut the Miniprep DNA along with the original DNA and some Lambda DNA using Restriction Enzymes. ( DNA Digest )
6. Separate the fragments by their size using Electrophoresis.
7. Compare the fragment sizes.
Electrophoresis
Fragments of DNA that Have been cut by
Restriction
Enzymes can be separated by
their size and their negative charge in an electric field.
The DNA is held
in a gel matrix.
How electrophoresis works
http://www.sumanasinc.com/webcontent/animat ions/content/gelelectrophoresis.html
Electrophoresis Gel from a Lambda/HindHI digest
This is the molecular size marker
used in your lab.
Southern Blot -DNA Finger Print
Western Blots separate and Identify
Proteins using Antibody Probes. Adding SDS makes all proteins negative.
Northern Blots separate RNA.
RNA and DNA
1. When you are working with large amounts of DNA, like from a person, you can look for specific genes or regions of DNA using a
hybridization probe of RNA or DNA.
2. These probes are radioactive or fluorescent so they can be seen.
3. DNA hybridization can be used in comparing the evolutionary
relatedness of organisms, or to
match the identity of an individual.
Some other applications of using DNA Hybridization
In doing a genetic finger print. There are 8 or more sites that are used in the identification of an individual in a court of law.
The sites that are used are found in everyone but with slight variation . People have two genes for each trait.
People can be
Heterozygous (Aa) Or
Homozygous (AA or aa) AA = 1 cut, so 2 restriction fragments
aa = no cut, 1 fragment Aa = both possibilities, 3 fragments
RFLP Keys in on Differences
Transposons are mobile genetic elements
Jump or copy/paste
DNA to DNA
Retrotransposons
always change BP #s
Copy / paste
DNA to RNA to DNA
# of Tandem repeats (STRs Simple Tandem Repeats) can also be unique
Chromosome Flyover:
http://www.dnalc.org/re sources/3d/chr11.html
Transposon
New copy of transposon
Transposon is copied DNA of genome
Insertion
Mobile transposon
(a) Transposon movement (“copy-and-paste” mechanism)
Retrotransposon
New copy of retrotransposon
DNA of genome
RNA Reverse
transcriptase
(b) Retrotransposon movement
Insertion
See p. 434-436
&
p. 448-450
RFLP’s
No 3rd cut site Genetic diversity
Repeats change The fragment sizes.
Variable Number Tandem Repeats, (STR’s)
PCR amplifies just the genes of interest.
D2’s father was the milk man, and S2 is the neighbor’s kid.
Children cannot have a band that neither parent has…
Another Application of DNA
Hybridization is to make DNA Chips.
Called Microarrays.
1. Coat chip with known genes
2. Pull mRNA from
study cell & convert to fluor single strand DNA
(reverse transcriptase) = cDNA
3. Flood chip, count hybrids
Diagnostics
Identification of a
shared or unique gene.
A DNA profile
http://www.dnalc.org/resources/3d/
26-microarray.html
Is the cDNA the same in all of your cells?
Even though all of your cells have the same
genes, different cells use different genes to make proteins unique to those cells.
Nerve cells make
different proteins than Liver cells and would then have different cDNA.
cDNA =
Coding (com
plimentary)
DNA, or DNA that
codes for a
protein.
Change the World?
Diagnosis
Evolution
Classification
Forensics
Agriculture, GM
Treatment
Sequencing DNA
There are several
ways to find the actual order of nucleotides in a strand of DNA.
One way is called the Sanger method.
Another cycle
sequencing
http://www.dnalc.org/d
dnalc/resources/sange
rseq.html
Sanger Method for DNA
Sequencing
DNA
Sequencing Automated
Each Dideoxy
nucleotide is given a fluorescent tag.
A = Green
C = Blue
G = Yellow
T = Red
All of the fragments are scanned.
http://www.dnalc.org/reso urces/animations/cycseq.
html
The Computer Records
the Order
Genomic libraries… as part of sequencing… using bacteria as a vector
