Tokyo/Works/Hybrid promoter
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__NOTOC__ | __NOTOC__ | ||
| - | <br>[[Tokyo/Works|Works top]] | + | <br>[[Tokyo/Works|Works top]] 0. [[Tokyo/Works/Hybrid promoter|Hybrid promoter]] 1. [[Tokyo/Works/Formulation |Formulation]] 2. [[Tokyo/Works/Assay |Assay1]] 3. [[Tokyo/Works/Simulation |Simulation]] 4. [[Tokyo/Works/Assay2 |Assay2]] 5. [[Tokyo/Works/Future works |Future works]] |
<br>[[Tokyo/Works/Assay0|Assay0]] | <br>[[Tokyo/Works/Assay0|Assay0]] | ||
| - | == | + | ==Objective== |
| - | [[Image:Hybrid.JPG|thumb|150px| '''Fig.1: Hybrid promoter'''<br>The newly | + | [[Image:Hybrid.JPG|thumb|150px| '''Fig.1: Hybrid promoter'''<br>The newly devised lux lac hybrid promoter is activated by AHL (exactly saying, AHL-LuxR complex) and repressed by LacI.|right]] |
To realize our model, expression of one part of the genes should be regulated by two factors. Therefore, the promoter upstream of the idlers genetic part was designed to '''sense TWO INPUTS, activated by AHL and repressed by LacI'''. | To realize our model, expression of one part of the genes should be regulated by two factors. Therefore, the promoter upstream of the idlers genetic part was designed to '''sense TWO INPUTS, activated by AHL and repressed by LacI'''. | ||
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Based on the AHL regulated BioBrick part R0062 and the previous work on LacI regulated promoter (ref. 1), we designed a new promoter regulated by AHL and LacI. Since this promoter is a kind of a '''"HYBRID"''' of the two, we call it '''HYBRID PROMOTER'''. | Based on the AHL regulated BioBrick part R0062 and the previous work on LacI regulated promoter (ref. 1), we designed a new promoter regulated by AHL and LacI. Since this promoter is a kind of a '''"HYBRID"''' of the two, we call it '''HYBRID PROMOTER'''. | ||
[[Image:Hybrid promoter.JPG|thumb|350px| '''Fig.2: Sequence of the lux lac hybrid promoter.''' <br>It contains luxR binding site called lux box, lacI binding sites O1, and -35 and -10 regions.|right]] | [[Image:Hybrid promoter.JPG|thumb|350px| '''Fig.2: Sequence of the lux lac hybrid promoter.''' <br>It contains luxR binding site called lux box, lacI binding sites O1, and -35 and -10 regions.|right]] | ||
| - | <br>Compared with existing BioBrick hybrid promoter R0065, | + | <br>Compared with existing BioBrick hybrid promoter R0065, it is repressed by LacI, not CI. In addition, there is no quantitative data for R0065 as usual Biobrick parts. Because we aimed to adjust effective concentration of a repressor for part characterization, CI is unfavorable to our project. We actually describe clear output pattern dependent on two inputs in Fig 3. We further analyzed detailed character of the parts as shown in [[Tokyo/Works/Assay |the latter section]]. In addition, this promoter is different from the previous one in that its sequence is much shorter with lux box and -35 region overlapping. |
<br><br>'''References''' | <br><br>'''References''' | ||
<br>-Biobrick R0062 | <br>-Biobrick R0062 | ||
| - | <br>-Lutz, R., and H. Bujard. 1997. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I-1-I-2 regulatory elements. Nucleic Acids Res. 25:1203–1210. | + | <br>-Lutz, R., and H. Bujard. 1997. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I-1-I-2 regulatory elements. ''Nucleic Acids Res.'' '''25''':1203–1210. |
==Experiments== | ==Experiments== | ||
| - | [[Image:Hybridgraph3.JPG|thumb|300px| '''Fig.3: The | + | [[Image:Hybridgraph3.JPG|thumb|300px| '''Fig.3: The effect of AHL and LacI on hybrid promoter''' <br>Only in the presence of AHL and in the absence of lacI, the lux lac hybrid promoter is activated significantly.|right]] |
The characteristics of this hybrid promoter (Lux-lac hybrid promoter) have been determined though '''[[Tokyo/Works/Assay0 |Assay 0]]'''. The results are shown in Fig.3. | The characteristics of this hybrid promoter (Lux-lac hybrid promoter) have been determined though '''[[Tokyo/Works/Assay0 |Assay 0]]'''. The results are shown in Fig.3. | ||
'''The newly devised hybrid promoter is activated by AHL and repressed by LacI.''' | '''The newly devised hybrid promoter is activated by AHL and repressed by LacI.''' | ||
Latest revision as of 05:40, 27 October 2007
Works top 0. Hybrid promoter 1. Formulation 2. Assay1 3. Simulation 4. Assay2 5. Future works
Objective
The newly devised lux lac hybrid promoter is activated by AHL (exactly saying, AHL-LuxR complex) and repressed by LacI.
To realize our model, expression of one part of the genes should be regulated by two factors. Therefore, the promoter upstream of the idlers genetic part was designed to sense TWO INPUTS, activated by AHL and repressed by LacI.
Design
Based on the AHL regulated BioBrick part R0062 and the previous work on LacI regulated promoter (ref. 1), we designed a new promoter regulated by AHL and LacI. Since this promoter is a kind of a "HYBRID" of the two, we call it HYBRID PROMOTER.
It contains luxR binding site called lux box, lacI binding sites O1, and -35 and -10 regions.
Compared with existing BioBrick hybrid promoter R0065, it is repressed by LacI, not CI. In addition, there is no quantitative data for R0065 as usual Biobrick parts. Because we aimed to adjust effective concentration of a repressor for part characterization, CI is unfavorable to our project. We actually describe clear output pattern dependent on two inputs in Fig 3. We further analyzed detailed character of the parts as shown in the latter section. In addition, this promoter is different from the previous one in that its sequence is much shorter with lux box and -35 region overlapping.
References
-Biobrick R0062
-Lutz, R., and H. Bujard. 1997. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I-1-I-2 regulatory elements. Nucleic Acids Res. 25:1203–1210.
Experiments
Only in the presence of AHL and in the absence of lacI, the lux lac hybrid promoter is activated significantly.
The characteristics of this hybrid promoter (Lux-lac hybrid promoter) have been determined though Assay 0. The results are shown in Fig.3.
The newly devised hybrid promoter is activated by AHL and repressed by LacI.
In the present project, this hybrid promoter has been tested on the last year's A-4 BioBrick part (BBa_J54140) by replacing with its original promoter lux pR (R0062).
Works top << Hybrid promoter >> Next(Formulation)
