Difference between revisions of "Part:BBa K142024"

Latest revision as of 00:21, 29 October 2008

IPTG-on tetracycline-off pulse generator with LacI mutant (R197A) and 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).

A LacI repressor tetramer, residues R197 and T276 are shown in red. B IPTG bound to the inducer binding site of the lac repressor, residues R197 and T276 are shown in green. Molecular graphics was generated from coordinate set [http://www.rcsb.org/pdb/explore.do?structureId=1LBH 1lbh] (1) using [http://www.cgl.ucsf.edu/chimera/ UCSF Chimera].

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1360
Illegal NheI site found at 1383
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 5: / Line 5: @@
 ===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.
+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).
+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).
 [[image:jr_pulsegen_1.jpg|frame|none|
-A Lac repressor tetramer, residues R197 and T276 are shown in red. B IPTG bound to the inducer binding site of the lac repressor, residues R197 and T276 are shown in green. Molecular graphics was generated from coordinate set [http://www.rcsb.org/pdb/explore.do?structureId=1LBH 1lbh] (1) using  [http://www.cgl.ucsf.edu/chimera/ UCSF Chimera].]]
+A LacI repressor tetramer, residues R197 and T276 are shown in red. B IPTG bound to the inducer binding site of the lac repressor, residues R197 and T276 are shown in green. Molecular graphics was generated from coordinate set [http://www.rcsb.org/pdb/explore.do?structureId=1LBH 1lbh] (1) using  [http://www.cgl.ucsf.edu/chimera/ UCSF Chimera].]]
 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.
@@ Line 36: / Line 37: @@
 ====Purpose of this BioBrick====
-This BioBrick is based on BioBrick [https://parts.igem.org/Part:BBa_I763026 I763026] in the original form. As sequencing by Caltech has shown that BioBrick [https://parts.igem.org/Part:BBa_I763026 I763026] did not have a promotor, we have added tetracycline-inducible promotor [https://parts.igem.org/Part:BBa_R0040 R0040] to the Biobrick and added TetR expression cassette [https://parts.igem.org/Part:BBa_I739001 I739001] froming device [https://parts.igem.org/wiki/index.php?title=Part:BBa_K142047 K142047] before subjecting it to site-directed mutagenesis. Expression of the mutant LacI IS (and silencing of lac-controlled gene expression) can therefore be initiated by addition of inducer tetracycline.
+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:===
@@ Line 45: / Line 46: @@
 (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.
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