TOPO Cloning Lecture

TOPO Cloning Lecture

1. A Brief Diversion Concerning Negative Regulation
from Lehninger Principles of Biochemistry, 4
th
ed.
Before we discuss TOPO cloning, we need some
background on the following:

2. Signal
molecule
in lac
operon is
allolactose

3. What happens when a
bacterium encounters
milk sugar
IPTG is a structural analog of
allolactose and will induce tn.
from Lehninger Principles of Biochemistry, 4th ed.

4. The French
scientists
who elucidated
the regulation
of the lac
operon
from Lehninger Principles of Biochemistry, 4th ed.

5. The lac operon
from Lehninger Principles of Biochemistry, 4th ed.

6. from Lehninger Principles of Biochemistry, 4th ed.
Note the
conformational
change in the
bound (solid)
vs. free (trans-
parent).

7. TOPO Cloning from Invitrogen

8. Other TOPO Vectors

11. About Stewart Shuman
Stewart Shuman is Professor of Molecular Biology
at the Memorial Sloan-Kettering Cancer Center,
New York, United States. He obtained his M.D.
and Ph.D. at the Albert Einstein College of Medicine,
New York, and completed a residency in Internal
Medicine at the Massachusetts General Hospital,
USA. He was a medical staff fellow in virology at
the National Institutes of Health before joining the
Sloan-Kettering faculty in 1998. Shuman's laboratory
is studying the mechanisms and structures of
enzymes that catalyze essential RNA and DNA
transactions. The laboratory is especially interested
in the divergence of enzyme mechanisms between
host and pathogen, and its implications for identifying
new targets for the treatment of infectious diseases.
http://www.nature.com/nrm/journal/v3/n8/authors/nrm880.html

14. CAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCT
GCCACCATACCCACGCCGAAACAAGCGCTC ATGAGCCCGA
AGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATA
TAGGCGCCAGCAACCGCAC CTGTGGCGCCGGTGATGCCGG
CCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCG
CGAAATTA ATACGACTCACTATAGGGGAATTGTGAGCGGATA
ACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAA
TTCAGGAGCCCTTCACCAAGGGCGAGCTCAATTCGAAGCTTG
AAGGTAAGCCTATCCCTAAC CCTCTCCTCGGTCTCGATTCTAC
GCGTACCGGTCATCATCACCATCACCATTGAGTTTGATCCGGC
TGCT…
The pET101D vector sequence

16. The area of interest