Difference between revisions of "Part:BBa K2442208"

Latest revision as of 22:47, 1 November 2017

Pmtle + B0034 + GFP This part contains the minimal pmtlE with added B0034 RBS to create a variant promoter element which was 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.

Usage and Biology

The mannitol operon exists naturally in P.fluorescens. 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 P.fluorescens. The minimal promoter was used in this construct in order to add a different RBS, (B0032). For this reporter construct pmtlE was ligated upstream of GFP, which would allow for fluorescent measurements.

Characterization

In order to test the working of our reporter constructs, these were ligated into E.coli, DH5a cells. We studied the induction of this promoter with mtlR with a number of different sugars, mannitol, sorbitol, sucrose, xylose, ribose, and fructose. The reporter plasmid variant was tested alone with these sugars as well as being tested alongside the regulatory plasmid.

Figure 1: GFP response to the sugar induction of regulatory and or reporter plasmid

Our results show no fluorescent response from either of the reporter plasmid variant alone, or once combined with the regulatory plasmid. However, in both cases there is a fluorescent response from the WT version of the reporter plasmid. These results suggest that the WT version of PmtlE is active and as shown by the two fold increase in GFP expression upon addition of the reporter plasmid, is inducible by mtlR. As the B0034 variant shows no fluorescent response, this suggests that the removal of some of the promoter region was vital in the induction of the promoter.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 869

@@ Line 10: / Line 10: @@
 In order to test the working of our reporter constructs, these were ligated into <i>E.coli</i>, DH5a cells. We studied the induction of this promoter with mtlR with a number of different sugars, mannitol, sorbitol, sucrose, xylose, ribose, and fructose. The reporter plasmid variant was tested alone with these sugars as well as being tested alongside the regulatory plasmid.
-[[Image:T-Glasgow-finalmtlrimage.png|450px|thumb|left|'''Figure 1:''' GFP response to the sugar induction of regulatory and or reporter plasmid]]
+[[Image:T-Glasgow-pleaseworkgraph.png|450px|thumb|left|'''Figure 1:''' GFP response to the sugar induction of regulatory and or reporter plasmid]]
 Our results show no fluorescent response from either of the reporter plasmid variant alone, or once combined with the regulatory plasmid. However, in both cases there is a fluorescent response from the WT version of the reporter plasmid. These results suggest that the WT version of PmtlE is active and as shown by the two fold increase in GFP expression upon addition of the reporter plasmid, is inducible by mtlR. As the B0034 variant shows no fluorescent response, this suggests that the removal of some of the promoter region was vital in the induction of the promoter.