Measurement

Part:BBa_K1316005

Designed by: Joan Cortada Garcia   Group: iGEM14_TU_Delft-Leiden   (2014-08-24)
Revision as of 12:44, 15 October 2014 by Joanctd.igem (Talk | contribs)

ybiJ promoter coupled to mKate2 reporter gene

Promoter of the ybiJ gene, which is activated by the presence of several nitrogen-based compounds such as 2,4,6-TNT, 2,4-DNT and 1,3-DNB. mKate2 is a far-red fluorescent protein used with reporting purposes. mKate2 codon usage is optimised for high expression in mammalian cells (humanised). It is, nevertheless, suitable for propagation in E. coli. This construct is, then, designed to be able to detect and quantify the presence of compunds such as 2,4,6-TNT, 2,4-DNT and 1,3-DNB.

Characterisation

Different concentrations of 2,4-DNT were used to test for induction of mKate2. The samples were analysed on the plate reader searching for fluorescence (Excitation wavelength: 588nm, Emission wavelength: 633nm). The BioBrick BBa_K1316005 (sample C on the Figures) showed an increasing fluorescent signal over time when induced with DNT but, consistently with the literature (Belkin et al., 2014), the threshold for BBa_K1316005 is bigger than for BBa_K1316003, hence, a higher concentration of 2,4-DNT is required to trigger a response for BBa_K1316005 than for BBa_K1316003. That can be observed in the lower response of BBa_K1316005 at 17mg/L of DNT. Besides, the intensity of the response is higher for BBa_K1316003 than for BBa_K1316005. When non-induced (0mg/L DNT), the constructs showed no clear increase in fluorescent signal. The non-induction of the negative control (sample D on the Figures) indicates that it is the presence of the promoter what generates the signal in front of the presence of DNT. Sample A is the positive control constitutively expressing mKate2.

<figure> <img src="TUDelft_2014_Few_induction.jpg" width="75%" height="75%"> <figcaption> Figure 1: Fluorescent signal emited by cells carrying constitutively expressed mKate2 (positive control), two paralel samples of the construct LD2 (BBa_K1316003) (Samples 1 and 2), and empty cells not carrying any BioBrick (negative control) 2 hours after induction (left) and 6 hours after induction (right). </figcaption> </figure>

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 381
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 886
    Illegal BsaI.rc site found at 1075


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