Composite

Part:BBa_K1682016

Designed by: iGEM15_HKUST-Rice   Group: iGEM15_HKUST-Rice   (2015-09-15)

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

HKUST-RICE BBa K1682016.1.jpg

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

Characterization

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.

Error creating thumbnail: Invalid thumbnail parameters

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 plate reader.

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 a 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]


[edit]
Categories
//chassis/prokaryote/ecoli
Parameters
None