Difference between revisions of "Part:BBa K2442206"
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This part contains the native [https://parts.igem.org/Part:BBa_K2442203 pmtlE promoter] ligated upstream of GFP [https://parts.igem.org/Part:BBa_E0040 (E0040)]. pmtlE is the intergenic region between mtlR and the mtlE coding region in <i>Pseudomonas Flourescens</i>. GFP is taken from the parts registry [https://parts.igem.org/Part:BBa_E0040 E0040]. This reporter plasmid was used in conjunction with a [https://parts.igem.org/Part:BBa_K2442202 regulatory plasmid] in order to induce the pmtlE. | This part contains the native [https://parts.igem.org/Part:BBa_K2442203 pmtlE promoter] ligated upstream of GFP [https://parts.igem.org/Part:BBa_E0040 (E0040)]. pmtlE is the intergenic region between mtlR and the mtlE coding region in <i>Pseudomonas Flourescens</i>. GFP is taken from the parts registry [https://parts.igem.org/Part:BBa_E0040 E0040]. This reporter plasmid was used in conjunction with a [https://parts.igem.org/Part:BBa_K2442202 regulatory plasmid] in order to induce the pmtlE. | ||
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The mannitol operon exists naturally in <i>P.fluorescens</i>. The regulatory protein in this operon, mtlR, binds with sugar to induce the mtlE promoter. This promoter has found to only be inducible from the binding of the mtlR protein-sugar complex. This system naturally induces the transcription of mannitol metabolism genes within <i>P.fluorescens</i>. For this reporter construct pmtlE was ligated upstream of GFP, which would allow for fluorescent measurements. | The mannitol operon exists naturally in <i>P.fluorescens</i>. The regulatory protein in this operon, mtlR, binds with sugar to induce the mtlE promoter. This promoter has found to only be inducible from the binding of the mtlR protein-sugar complex. This system naturally induces the transcription of mannitol metabolism genes within <i>P.fluorescens</i>. For this reporter construct pmtlE was ligated upstream of GFP, which would allow for fluorescent measurements. | ||
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===Characterization=== | ===Characterization=== | ||
In order to test the working of our reporter constructs, these were ligated into <i>E.coli</i>, DH5a cells. We studied the indiction of this promoter with mtlR with a number of different sugars, mannitol, sorbitol, sucrose, xylose, ribose, and fructose. The reporter plasmid was tested alone with these sugars as well as being tested alongside the regulatory plasmid. | In order to test the working of our reporter constructs, these were ligated into <i>E.coli</i>, DH5a cells. We studied the indiction of this promoter with mtlR with a number of different sugars, mannitol, sorbitol, sucrose, xylose, ribose, and fructose. The reporter plasmid was tested alone with these sugars as well as being tested alongside the regulatory plasmid. | ||
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<partinfo>BBa_K2442206 short</partinfo> | <partinfo>BBa_K2442206 short</partinfo> | ||
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<partinfo>BBa_K2442206 SequenceAndFeatures</partinfo> | <partinfo>BBa_K2442206 SequenceAndFeatures</partinfo> | ||
Revision as of 21:52, 1 November 2017
This part contains the native pmtlE promoter ligated upstream of GFP (E0040). pmtlE is the intergenic region between mtlR and the mtlE coding region in Pseudomonas Flourescens. GFP is taken from the parts registry E0040. This reporter plasmid was used in conjunction with a regulatory plasmid in order to induce the pmtlE.
Characterization
In order to test the working of our reporter constructs, these were ligated into E.coli, DH5a cells. We studied the indiction of this promoter with mtlR with a number of different sugars, mannitol, sorbitol, sucrose, xylose, ribose, and fructose. The reporter plasmid was tested alone with these sugars as well as being tested alongside the regulatory plasmid.
Our results show a fluoresence response both from the reporter plasmid alone as well as in addition with the regulatory plasmid. This suggests that mtlE within this construct is constitutively active. However, upon addition with the regulatory plasmid, there is almost a two fold increase in GFP response, suggesting the regulatory protein still contributes to the induction of pmtlE.
Pmtle + RBS WT + GFP
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 829