PROJECT A

Cloning the lac operon

Keywords: lac operon, regulation of transcription, lactose, repression, induction, transcription, IPTG, X-gal, glucose, E. coli.

Overview: The molecular details of how gene expression is controlled is usually addressed with genetic and biochemical methods. The first and the now well understood lac operon in E. coli has become the paradigm for gene regulation. In addition, several elements of the lac operon are widely used in the construction of molecular tools that are routinely used in most modern biotechnology research laboratories. Thus, lac DNA is rescuable from a wide range of vectors and powerful genetic E. coli backgrounds are available and ready to use. The lac operon includes two metabolic genes, Y and Z, that are required for metabolism of lactose (see Figure). The lacY encodes a lactose permease that is inserted into the cell membrane and is responsible for the transport of lactose across the membrane. LacZ encodes beta-galactosidase that splits lactose into glucose and galactose. In addition, the lac operon encodes an thiogalactoside transacetylase whose function is not well understood. beta-galacosidase and lactose permease are easily assayed and a large selection of specific substrates are available (i.e., X-gal). The lac regulon includes the promoter and lacI the gene that encodes a protein that represses operon transcription. Without lactose and presence of glucose (low cAMP levels) transcription is strongly repressed, cryptic transcription is observed in mixtures containing glucose and lactose and transcription is heavy in situations where only lactose (high cAMP levels) is present.

 

Goal: The objective of this laboratory assignment is to isolate, characterize and demonstrate the cloning of a complete and functional DNA copy of the entire E. coli lac operon. Cloning strategies and the genetic and biochemical proof are at the discretion of each group. There are no limitations, as long as your method isolates and identifies the DNA fragment of interest and the material resources are available.

Recommended Reading:

(***** Easy Reading (very useful); *** Classic Paper; * Important Contribution)

 

Gene Structure, Function and Regulation of Expression:

 

*** 1 - Beckwith, J. R. and D. Zipser, Eds. (1970). The lactose operon. Cold Spring Harbor Laboratory. New York, CSHL Press.

 

*** 2 - Jacob, F. and J. Monod (1961). Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol 3: 2415-2421.

 

***** 3 - Lodish, H., D. Baltimore, A. Berk, L. Zipursky, P. Matsudaira and J. Darnell (1995). Chapter 9: The molecular anatomy of genes and chromosomes. Molecular Cell Biology Ed. t. edition. New York, W.H. Freeman and Company. 307-364.

 

***** 4 - Lodish, H., D. Baltimore, A. Berk, L. Zipursky, P. Matsudaira and J. Darnell (1995). Chapter 11: Regulation of transcription initiation. Molecular Cell Biology Ed. t. edition. New York, W.H. Freeman and Company. 405-480.

 

* 5 - Malan, T. P. and W. R. McClure (1984). Dual promoter control of the Escherichia coli lactose operon. Cell 39: 173 180.

 

*** 6 - Monod, J. (1966). From enzymatic adaptation to allosteric transitions. Science 154: 475 483.

 

* 7 - Oehler, S., M. Amouyal, P. Kolkhof, B. von W. Bergmann and B. M. Hill (1994). Quality and position of the three lac operators of E. coli define efficiency of repression. Embo J 13: 3348 3355.

 

*** 8 - Pardee, A. B., F. Jacob and J. Monod (1959). The genetic control and cytoplasmic expression of inducibility in the synthesis of b-galactosidase by E. coli.1: 165-178.

 

* 9 - Reznikoff, W. (1992). Catabolite gene activator protein activation of lac transcription. J Bacteriol 174: 655-658.

 

*** 10 - Simpson, R. B. (1980). Interaction of the cAMP receptor protein with the lac promoter. Nucleic Acids Res 8: 759 766.

 

*** 11 - Spassky, A., S. Busby and H. Buc (1984). On the action of the cyclic AMP cyclic AMP receptor protein complex at the Escherichia coli lactose and galactose promoter regions. Embo J 3: 43 50.

 

* 12 - Ullmann, A., E. Joseph and A. Danchin (1979). Cyclic AMP as a modulator of polarity in polycistronic transcriptional units. Proc Natl Acad Sci U S A 76: 3194 3197.

 

*** 13 - Ullmann, A., F. Tillier and J. Monod (1976). Catabolite modulator factor: a possible mediator of catabolite repression in bacteria. Proc Natl Acad Sci U S A 73: 3476 3479.

 

Reference to Methods and Molecular Biology Handbooks:

 

14 - Ausubel, F. M. (1987). Current Protocols in Molecular Biology. Wiley.

 

15 - Glick, B. R. and J. J. Pasternak (1994). Chapter 1: The molecular biology revolution. Molecular Biotechnology: Principles and applications of recombinant DNA . Washington D.C., ASM Press. 5-16.

 

16 - Glick, B. R. and J. J. Pasternak (1994). Chapter 2: Recombinant DNA technology. Molecular Biotechnology: Principles and applications of recombinant DNA . Washington D.C., ASM Press. 17-54.

 

17 - Glick, B. R. and J. J. Pasternak (1994). Chapter 3: Molecular research procedures. Molecular Biotechnology: Principles and applications of recombinant DNA . Washington D.C., ASM Press. 55-82.

 

18 - Glick, B. R. and J. J. Pasternak (1994). Chapter 4: Manipulation of gene expression in prokaryotes. Molecular Biotechnology: Principles and applications of recombinant DNA . Washington D.C., ASM Press. 83-112.

 

19 - Lodish, H., D. Baltimore, A. Berk, L. Zipursky, P. Matsudaira and J. Darnell (1995). Chapter 7: Recombinant DNA Technology. Molecular Cell Biology Ed. t. edition. New York, W.H. Freeman and Company. 221-260.

 

20 - Lodish, H., D. Baltimore, A. Berk, L. Zipursky, P. Matsudaira and J. Darnell (1995). Chapter 8: Genetic analysis in cell biology. Molecular Cell Biology Ed. t. edition. New York, W.H. Freeman and Company. 263-303.

 

21 - Primrose, S. B. (1991). Part II: Recombinant DNA technology. Molecular Biotechnology . Oxford, UK, Blackwell Scientific Publications. second edition, ed. 13-57.

 

22 - Primrose, S. B. (1991). Part III: The exploitation of microbes. Molecular Biotechnology . Oxford, UK, Blackwell Scientific Publications. second edition, ed. 61-108.

 

23 - Sambrook, J., E. F. Fritsch and T. Maniatis (1989). Molecular Cloning. New York, CSHL Press.

 

Ethics and Regulatory Issues in Biotechnology

 

24 - Glick, B. R. and J. J. Pasternak (1994). Chapter 18: Regulating the use of biotechnology. Molecular Biotechnology: Principles and applications of recombinant DNA . Washington D.C., ASM Press. 425-441.

 

25 - Glick, B. R. and J. J. Pasternak (1994). Chapter 19: Patenting biotechnology inventions. Molecular Biotechnology: Principles and applications of recombinant DNA . Washington D.C., ASM Press. 443-450.