Difference between revisions of "Part:BBa K1316003"

Revision as of 09:27, 6 October 2014

yqjF promoter coupled to mKate2 reporter gene

Promoter of the yqjF 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_K1316003 (sample B on the Figures) showed an increasing fluorescent signal over time when induced with DNT. When non-induced (0mg/L DNT), the constructs showed no clear increase in fluorescent signal. The BioBrick BBa_K1316003 showed a much higher response than BBa_K1316005 (sample C on the Figures), hence, the yqjF promoter responds better to DNT than the ybiJ promoter, consistently with the literature (Belkin et al., 2014). At the same time, BBa_K1316003 is more sensitive to 2,4-DNT than BBa_K1316005 showing the first one a lower threshold for 2,4-DNT than the second one. 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. Samples: A - Positive control (Constitutive mKate2) B - BBa_K1316003 C - BBa_K1316005 D - Negative control (cells without the construct)

Sequence and Features