Part:BBa_K142028
IPTG-on tetracycline-off pulse generator with LacI mutant (R197A, T276A)/TetR expression cassette
IPTG-on/tetracycline-off switch circuit
Short description:
This part contains a tetracycline-controlled LacI IS expression cassette followed by a constitutive TetR expression cassette. The LacI IS mutant is almost identical to the LacI transcriptional regulator except for the difference that it is not able to bind IPTG or allolactose due to a mutation; it therefore can not be activated by induction with these substances. Since it recognizes the same motif in the lac promotor region, it strongly represses transcription of all genes regulated by promotors with lacI binding site even if IPTG or allolactose are present. It is used to terminate the expression of proteins under lac control if IPTG can not be removed from the cell rapidly.
Detailed description:
LacI IS mutants are uninducible repressors of lac-controlled promotors
Expression of the lac operon in E. coli is tightly controlled by LacI, a protein, which binds to a repressor binding site within the promotor and disables transcription by obscuring the promotor region. When bound to DNA, LacI is in the tetrameric form, which consists of two dimers interacting at the end distal from the DNA binding site. Upon binding of allolactose or IPTG, the tetramer breaks down into two dimers and the affinity for the repressor binding site is greatly reduced; the lacI IPTG complex will diffuse away from the repressor binding site, leaving the promotor accessible. As a result of decades of genetic and structural studies, the function of lacI is now understood on the molecular level (1, 2). Mutational experiments have identified residues, which abolish IPTG response upon mutation (3). Furthermore, the x-ray crystal structure of LacI with bound IPTG has allowed the identification of residues that interact with IPTG and which are promising targets for mutagenesis (1).
We decided to mutate residues R197 and T276, which are located in the IPTG binding groove, contact IPTG and have been shown to produce the lacI IS mutation in previous genetic experiments. We decided to generate a set of eight mutated lacIs, in which we replaced either R197 with alanine or phenylalanine or T276 with alanine or phenylalanine or both in all possible combinations.
lacIIS-1: R197A
lacIIS-2: R197F
lacIIS-3: T276A
lacIIS-4: T276F
lacIIS-5: R197A T276A
lacIIS-6: R197A T276F
lacIIS-7: R197F T276A
lacIIS-8: R197F T276F
All mutants appeared to be equally efficient in preliminary genetic experiments with full repression even at elevated concentrations of 10mM IPTG. Please see the chapter on the switch circuit in the [http://2008.igem.org/Team:ETH_Zurich/Wetlab/Switch_Circuit ETH 2008 Wiki] for details.
Purpose of this BioBrick
This BioBrick allows pulsing of protein expression under lac-controlled promotors. It is especially useful if change of medium to remove the inducer is not an option (such as inside a fermenter/chemostat). Since the signal for stop of expression is independent from the induction of expression, pulses of different duration can be tested without any modification of the pulse generator construct.
References:
(1) Lewis, M., Chang, G., Horton, N. C., Kercher, M. A., Pace, H. C., Schumacher, M. A., Brennan, R. G., and Lu, P. (1996) Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Science 271, 1247-54.
(2) Friedman, A. M., Fischmann, T. O., and Steitz, T. A. (1995) Crystal structure of lac repressor core tetramer and its implications for DNA looping. Science 268, 1721-7.
(3) Suckow, J., Markiewicz, P., Kleina, L. G., Miller, J., Kisters-Woike, B., and Muller-Hill, B. (1996) Genetic studies of the Lac repressor. XV: 4000 single amino acid substitutions and analysis of the resulting phenotypes on the basis of the protein structure. J Mol Biol 261, 509-23.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1360
Illegal NheI site found at 1383 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |