Difference between revisions of "Part:BBa K2116025"

Latest revision as of 18:11, 25 October 2016

EsaR repressible promoter

An esabox is an 18bp sequence to which the transcriptional regulator EsaR Part:BBa_K2116001 can bind. We placed one esabox right after the normally constitutive Anderson promoter Part:BBa_J23118 to create an EsaR repressible promoter (see Figure 1). Transcription can be initiated by the specific AHL EsaR responds to [N-(3-oxo-hexanoyl)-L-homoserine lactone].

Figure 1: An esabox can be placed downstream of a promoter, effectively acting as a roadblock by binding EsaR.

When characterising this part, we used the D91G variant of EsaR, which has been documented to be more responsive to lower AHL concentrations compared to the wild type EsaR [1]. This EsaR variant was obtained through addgene (Plasmid #47646), and was also added to the registry Part:BBa_K2116001.

Two copies of EsaR were placed under the control of individual constitutive promoters Part:Bba_J23118 and expressed on a medium-low copy plasmid (replication origin pBR322/rop, kanamycine resistance). Based on the 2013 paper by Shong&Collins we decided to test AHL concentrations in the range of 0 to 10 µM, since in all combinations of esabox placements tested in this paper showed saturation at 10 µM.

We observed EsaR responds to the same AHL, but did not see the same saturation behaviour. We suggest that the saturation is highly dependent on the amount of EsaR and it was therefore tried to vary it by expressing EsaR on plasmids with different oris and just one copy of EsaR. A more detailed discussion can be found on [http://2016.igem.org/Team:ETH_Zurich/LabResults]

Figure 2: Dose response curve of EsaR. Induction with AHL at given concentrations. A dose response is observed, a saturation, however, is absent.

References:

[1] Shong, Jasmine and Cynthia H. Collins. "Engineering The Esar Promoter For Tunable Quorum Sensing-Dependent Gene Expression". ACS Synth. Biol. 2.10 (2013): 568-575.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2116025 short</partinfo>
-An esabox is an 18bp sequence to which the transcriptional regulator EsaR can bind. We placed one esabox right after the normally constitutive Anderson promoter [Bba_J23118] to create an EsaR repressible promoter. Transcription can be initiated by the specific AHL EsaR responds to [N-(3-oxo-hexanoyl)-L-homoserine lactone].
+An esabox is an 18bp sequence to which the transcriptional regulator EsaR [[Part:BBa_K2116001]] can bind. We placed one esabox right after
+the normally constitutive Anderson promoter [[Part:BBa_J23118]] to create an EsaR repressible promoter (see Figure 1). Transcription can be
+initiated by the specific AHL EsaR responds to [N-(3-oxo-hexanoyl)-L-homoserine lactone].
-When characterising this part, we used the D91G variant of EsaR, which has been documented to be more responsive to lower AHL concentrations compared to the wild type EsaR [1]. This EsaR variant was obtained through addgene, and can be found on the registry [BBa_K2116001].
+[[File:T--ETH_Zurich--esaboxplacement.png|200px|thumb|right|<b>Figure 1:</b> An esabox can be placed downstream of a promoter, effectively acting as a roadblock by binding EsaR.]]
-Two copies of EsaR were placed under the control of individual constitutive promoters [Bba_J23118] and expressed on a medium-low copy plasmid (replication origin pBR322/rop). Based on the 2013 paper by Shong&Collins we decided to test AHL concentrations in the range of 0 to 10 000nM, since in all combinations of esabox placements tested in this paper showed saturation at 10 000nM as seen below:
+When characterising this part, we used the D91G variant of EsaR, which has been documented to be more responsive to lower AHL
+concentrations compared to the wild type EsaR [1]. This EsaR variant was obtained through addgene (Plasmid #47646), and was also added to the registry [[Part:BBa_K2116001]].
-[[File:BBa K2116025 Collinsdata.png|200px|frame|center|upright=0.2|Dose response curves of EsaR, tested on promoters with various esabox placements. (Shong&Collins 2013 [1]).]]
+Two copies of EsaR were placed under the control of individual constitutive promoters [[Part:Bba_J23118]] and expressed on a
+medium-low copy plasmid (replication origin pBR322/rop, kanamycine resistance). Based on the 2013 paper by Shong&Collins we decided to test AHL
+concentrations in the range of 0 to 10 µM, since in all combinations of esabox placements tested in this paper showed saturation at 10 µM.
-We could not observe the same saturation behaviour in our tests,
+We observed EsaR responds to the same AHL, but did not see the same saturation behaviour. We suggest that the saturation is highly dependent on the amount of EsaR
+and it was therefore tried to vary it by expressing EsaR on plasmids with different oris and just one copy of EsaR. A more detailed discussion can be found on [http://2016.igem.org/Team:ETH_Zurich/LabResults]
+[[File:BBa_K2116025_EsaR_dosecurve.png|500px|thumb|center|<b>Figure 2:</b> Dose response curve of EsaR. Induction with AHL at given concentrations. A dose response
+is observed, a saturation, however, is absent.]]
-[[File:BBa_K2116025_EsaR_dosecurve.png|200px|thumb|center|Dose response curve of EsaR.]]
@@ Line 19: / Line 28: @@
 References:
 [1] Shong, Jasmine and Cynthia H. Collins. "Engineering The Esar Promoter For Tunable Quorum Sensing-Dependent Gene Expression". ACS Synth. Biol. 2.10 (2013): 568-575.