Difference between revisions of "Part:BBa K566002"

Latest revision as of 19:03, 28 September 2011

Biphasic switch

The biphasic switch combines positive and negative regulation through a single input. It is turned ON by low lambda cI concentrations and OFF by high cI concentrations(1), behavior shown in figure 1. The gene of interest to be controlled by the biphasic switch must be placed between pRM (BBa_K566001) and OL region (BBa_K566000), under the control of pRM promoter.

Figure 1. The Biphasic Switch. Behavior of wild type (wt) Lambda’s pRM in presence of the OL region placed 3.8 kb apart for this assay. LacZ gene is placed under pRM’s control. At low concentrations of cI, lacZ expression is stimulated. At high concentrations of cI, lacZ gets repressed. Figure taken and modified from Dodd BI et al. (2001) Gene Dev 15:3013–3022.

Usage and Biology

The gene of interest to be controlled through the Biphasic Switch must be placed between pRM and OL region, under the control of pRM promoter. Both repression and expression of the gene should be achieved through varying lambda cI protein concentration; strategy then consists on placing cI (BBa_K327018) under an inducible promoter and varying the stimuli to which the promoter responds. Low input would induce expression from pRM while high input would repress it.

There are two well-known sets of cI binding sites in lambda (OR and OL) spaced 2.4 kb apart and composed of three operators each (OR1:OR2:OR3; OL1:OL2:OL3) (2). cI has a higher affinity for OR1 and OR2 operators, where binding positively regulates the pRM promoter. In presence of the OL set, cI octamerise specifically binding to the two sets of operators (OR and OL) at the same time and therefore forming a DNA loop (3) as shown in figure 2. Such structure stabilizes cI's binding to OR3, allowing pRM's repression at high cI concentrations.

Figure 2. (a) EM picture of a circular relaxed plasmid with a large DNA loop formed by cI. Two tetramers on each DNA circle interact to form an octamer and thereby loop the DNA into a structure resembling a figure of eight. (b)Close up cartoon representing cI octamers occupying the OL1, OL2, OR1, and OR2 operators. OR3 is to be occupied as [cI] increases. The lower portion shows the sequence of PRM and OR3, the latter between the -10 and -35 where cI cooperative binding inhibits transcription. Figures taken and modified from: Révet B et al. (1999) Curr. Biol. 9: 151–154 and Dodd IB et al.(2001) Genes Dev 15:3013–3022, respectively.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 95
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K566002 short</partinfo>
-The biphasic switch combines positive and negative regulation through a single input. It is turned ON by low lambda cI concentrations and off by high cI concentrations ([https://parts.igem.org/Part:BBa_K566002:Design 1]). There are two well-known sets of cI binding sites in lambda (OR and OL) spaced 2.4 kb apart and composed of three operators each (OR1:OR2:OR3; OL1:OL2:OL3) ([https://parts.igem.org/Part:BBa_K566002:Design 2]). cI has a higher affinity for OR1 and OR2 operators, where binding positively regulates the pRM promoter. In presence of the OL set, cI octamerise specifically binding to the two sets of operators (OR and OL) at the same time and therefore forming a DNA loop ([https://parts.igem.org/Part:BBa_K566002:Design 3]). Such structure stabilizes cI's binding to OR3, allowing pRM's repression at cI high concentrations.
+The biphasic switch combines positive and negative regulation through a single input. It is turned ON by low lambda cI concentrations and OFF by high cI concentrations([https://parts.igem.org/Part:BBa_K566002:Design 1]), behavior shown in figure 1. The gene of interest to be controlled by the biphasic switch must be placed between pRM ([https://parts.igem.org/Part:BBa_K566001 BBa_K566001]) and OL region ([https://parts.igem.org/Part:BBa_K566000 BBa_K566000]), under the control of pRM promoter.
+ [[Image:Biphasic1.png|thumb|center|500px|'''Figure 1. The Biphasic Switch.''' Behavior of wild type (wt) Lambda’s pRM in presence of the OL region placed 3.8 kb apart for this assay. LacZ gene is placed under pRM’s control. At low concentrations of cI, lacZ expression is stimulated. At high concentrations of cI, lacZ gets repressed. Figure taken and modified from Dodd BI ''et al.'' (2001) ''Gene Dev'' '''15''':3013–3022.]]
 ===Usage and Biology===
-The biphasic switch may be used to control both expression and repression of pRM through a single input, which must control cI protein concentration. If repressor cI434 is placed under pRM's control along with the gene of interest, this switch may be coupled with the modified promoter pRM434 (which is stimulated by cI but repressed by cI434 [https://parts.igem.org/Part:BBa_I12006 BBa_I12006]) to form a two-state switch. The expected behavior will be that at low concentrations of cI, both promoters will be activated. However, because of cI434 repressing pRM434, only pRM will stay ON eventually leading to the first state. At high concentrations of cI, pRM -and consequently cI434- will be turned OFF, allowing pRM434 to be activated by cI and leading to a second state. pRM promoter from Phage 434 (not to confuse with pRM434 mentioned above) seems to exhibit the same biphasic behavior ([https://parts.igem.org/Part:BBa_K566002:Design 4]).
+<p>The gene of interest to be controlled through the Biphasic Switch must be placed between pRM and OL region, under the control of pRM promoter. Both repression and expression of the gene should be achieved through varying lambda cI protein concentration; strategy then consists on placing cI ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K327018 BBa_K327018]) under an inducible promoter and varying the stimuli to  which the promoter responds. Low input would induce expression from pRM while high input would repress it.</p>
+<p>There are two well-known sets of cI binding sites in lambda (OR and OL) spaced 2.4 kb apart and composed of three operators each (OR1:OR2:OR3; OL1:OL2:OL3) ([https://parts.igem.org/Part:BBa_K566002:Design 2]). cI has a higher affinity for OR1 and OR2 operators, where binding positively regulates the pRM promoter. In presence of the OL set, cI octamerise specifically binding to the two sets of operators (OR and OL) at the same time and therefore forming a DNA loop ([https://parts.igem.org/Part:BBa_K566002:Design 3]) as shown in figure 2. Such structure stabilizes cI's binding to OR3, allowing pRM's repression at high cI concentrations. </p>
+ [[Image:LOOP.png|thumb|center|700px|'''Figure 2'''. (a) EM picture of a circular relaxed plasmid with a large DNA loop formed by cI. Two tetramers on each DNA circle interact to form an octamer and thereby loop the DNA into a structure resembling a figure of eight. (b)Close up cartoon representing cI octamers occupying the OL1, OL2, OR1, and OR2 operators. OR3 is to be occupied as [cI] increases. The lower portion shows the sequence of PRM and OR3, the latter between the -10 and -35 where cI cooperative binding inhibits transcription. Figures taken and modified from: Révet B ''et al.'' (1999) ''Curr. Biol.'' '''9''': 151–154 and Dodd IB ''et al.''(2001) ''Genes Dev'' '''15''':3013–3022, respectively.]]
 <span class='h3bb'>Sequence and Features</span>