Difference between revisions of "Part:BBa K1682016"

Revision as of 04:31, 17 September 2015

IPTG induced RFP reporter system

HKUST-RICE team designed an IPTG inducible RFP reporter system. This was achieved by combining the constitutive promoter BBa_J23101 with LacI generator BBa_P0412, and a RFP coding device (BBa_J04450) driven by Plac.

Mechanism

Figure 1. Schematic diagram of the inducible Plac-mRFP construct. (a)In the absence of Isopropyl b-D-1-thiogalactopyranoside (IPTG), LacI represses Plac. (b)When IPTG is present, LacI protein is repressed, triggering the production of mRFP.

Usage and Functionality

In order to check the functionality of the construct for use in sensing IPTG concentrations, characterisation was done inducing the cell containing the BBa_K1682016 construct in pSB3K3 under varing IPTG concentrations.

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Figure 2. Dose response of E. coli DH10B containing pSB3K3-BBa_K1682016 to varing IPTG levels. After overnight induction of cell culture incubated in M9-glycerol at 37˚C, measurements for population averaged assay were taken by fluorometry.

The results from characterisation show that the working concentration of the constructed IPTG sensor is at around 0.001 to 0.5 mM IPTG, with an 4.5 fold dynamic range.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1084
Illegal NheI site found at 1107
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 2285
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 781
Illegal AgeI site found at 893
1000
COMPATIBLE WITH RFC[1000]

@@ Line 17: / Line 17: @@
 [[File:HKUST-RICE BBa K1682016 fig2.jpg|500px|centre|]]
-'''Figure 2. Dose response of <i>E. coli</i> DH10B containing pSB3K3-BBa_K1682016 to varing IPTG levels.''' After overnight induction of cell culture incubated in M9-glycerol at 37˚C, measurements were taken for population averaged assay by fluorometry.
+'''Figure 2. Dose response of <i>E. coli</i> DH10B containing pSB3K3-BBa_K1682016 to varing IPTG levels.''' After overnight induction of cell culture incubated in M9-glycerol at 37˚C, measurements for population averaged assay were taken by fluorometry.
 The results from characterisation show that the working concentration of the constructed IPTG sensor is at around 0.001 to 0.5 mM IPTG, with an 4.5 fold dynamic range.