Transcript Bacterial DNA Insert

BIOL 417 – Week 2, Day 2
•Prepared in large part by
–Celithelma Garcia
–Mijin Sohn and
–Flora Hwee of PGM 2000
•Revised 2012, SBSharp
Recap
1. Modification of the Insert:
When the cDNA is made, overhangs may
remain.
3’CCCCC---------------------------TTTT 5’
5’ GGGG---------------------------AA 3’
Add T4 polymerase and dNTPs:
3’ CCCC ---------------------------TTTT 5’
5’ GGGG---------------------------AAAA3’
Recap (continued)
2. PstI adaptors were added in excess to
polished cDNA to create sticky ends.
3. Ligase was added to connect the adaptor and
cDNA, thus completing the insert preparation.
4. Vector had been opened with PstI.
What’s next?
I. Forming recombinant plasmid DNA, i.e., the
“construct” (in vitro)
II. Transformation of ligation reaction products into
E. coli bacteria (in vitro  in vivo)
At least some of the ligation products are expected to be
vector + insert ligated together.
III. Selection of bacteria that have taken up plasmid
+/- insert (in vivo)
I. Forming the Recombinant
Construct
• The insert is added to the vector and ligase is
also added
3’ HO-ACGTC-----CCCCC--------------TTTTT------G-OH 5’
5’ HO-G-----GGGGG-------------AAAAA-----CTGCA-OH 3’
+
3’ HO-ACGTC-----------------------------------------------G-PO4 5’
5’ 4OP-G------------------------------------------------CTGCA-OH 3’
+
ligase
Diagram the ligation
product of the two
molecules on the prior
slide.
I. Recombinant (continued)
• Ligase connects where there is a -PO4-2 at
5’ end and –OH at adjacent 3’ end.
• Because the PstI adaptor’s 5’ sticky-end
has a chemically synthesized –OH group
rather than -PO4 -2 , ligase cannot ligate the
sticky end of the insert to the 3’ end –OH of
the vector.
• But, the construct remains together due to
the non-covalent H-bonds at the base pairs
throughout the length of the insert.
II.Transformation
Putting construct into bacteria for replication
• Definition: The process of introducing the
products of a ligation reaction into E. coli for
replication to high copy number.
Transformation
• The basic methodology is:
– Incubate E.coli in CaCl2 solution to make them
“competent”.
– Add ligation reaction to the competent E. coli.
– CaCl2 precipitates DNA into clumps and increases
DNA adsorption to cell membrane.
– Heat shock the bacteria/DNA mixture to facilitate
DNA uptake by the competent bacteria.
• Note: This same methodology is used with a
previously made plasmid construct in order to
grow up more of it.
II. Transformation
Possibilities of DNA uptake by bacteria (1).
1.
• Some bacteria take up reclosed
vector.
Bacterial
DNA
Vector
II. Transformation
Possibilities of DNA uptake by bacteria (2).
• Some bacteria take up only the
insert.
Bacterial
DNA
Insert
II. Transformation
Possibilities of DNA uptake by bacteria (3).
• Some bacteria take up the
product of interest, the
construct you want.
Bacterial
DNA
Vector + Insert
(recombinant)
II. Transformation
Possibilities of DNA uptake by bacteria (4).
• Some bacteria will contain a
construct with a shorter or a
longer insert than the one you
expect.
Bacterial
DNA
Vector + Shorter Insert
(recombinant)
IV. Possibilities in Attempting
to Transform Bacteria (5)
• Most bacteria will fail to take up
any ligation products.
Bacterial
DNA
Possibilities in Attempting to
Transform Bacteria (summary)
• There are more transformation
possibilities; only five are presented
1
2
3
4
5
•All types and more are in the same transformation reaction.
•We must distinguish bacteria that have taken up plasmid.
•Later, we must distinguish the product of interest from
other transformation products.
II. Summary: Putting construct into
bacteria for replication
(to get enough DNA to work with!)
• E. coli that have been made
competent can take up naked DNA.
• Only 1 in a 1000 competent E. coli
will take up DNA.
• Only a fraction of that fraction will
take up the recombinant construct
you want, because the construct
you want is only a fraction of
possible ligation products.
Realities of cDNA
construction.
• Some cDNAs will form that are not
full length.
• Some vectors will reclose on
themselves with no cDNA insert.
• Some vector molecules may never
have opened!
Nothing ever works the way you want
it to 100% of the time.
III. Selection of bacteria that have
taken up plasmid.
• How can you discriminate between bacteria that have
taken up plasmid (either +/- insert) and the other
99.9% of the bacteria?
– Take advantage of the drug resistance gene in the
plasmid vector.
• Ampr = gene that codes for an enzyme that breaks down
ampicillin, a drug that stops bacterial cell division.
• Because the plasmid includes a gene for Ampr, the
presence of the plasmid gives (confers) ampicillin
resistance to the bacterium.
• Include ampicillin in the nutrient agar in the bacterial
culture plate.
Restriction Map of Vectors:
pBR322 & pGEM®-3Z
Vector
IV. Selection of Desired
Recombinant Bacteria (cont.)
Transfer to a medium
without
ampicillin
Amp-
-Contains the Ampr gene
Transfer to a medium
with Amp
Transformed bacteria
<1/1000 have plasmid.
There are many
thousands of bacteria
in the tube.
Amp+
Notice the colonies.
The bacteria in each colony contain plasmids.
What must be present in the agar to ensure the
Summary of selection.
• All bacteria can grow on Amp- plate.
• Only those bacteria carrying
plasmids with an AmpR gene will
grow on Amp+ medium and form
colonies.
– Cells containing only the insert will not
grow on Amp+ medium.
Characteristics of a Good Vector
1 Origin of Replication: allows the vector to
replicate independently
2 Replicates to high copy number
3 Small genome that can be isolated easily
4 Unique restriction enzyme sites for DNA
insertion
5 A way to discriminate its presence in the
bacterium, e.g. ampicillin resistance
6 Optional: A way to discriminate the presence
of an insert, e.g. breakdown of X-gal by galactosidase (next time)