Difference between revisions of "Part:BBa K1316003"
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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. | 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. | 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. | ||
Revision as of 12:51, 15 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 type of experiments were conducted to characterize this BioBrick:
Plate Reader
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 ("pF" 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 ("pJ" 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 ("negative control" 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.
FACS
Using Fluorescence-activated cell sorting (FACS) technology we managed to see that inducing with DNT our best performing BioBrick of the Landmine detection module (BBa_K1316003) makes the cells carrying it generate an increase of fluprescence corresponding to mKate2 (Emission wavelenght: 633nm). The FACS technology allows us to see that, per cell, more fluorescence is produced by the construct BBa_K1316003 after several hours of their induction with DNT.
For more information about the characterisation of this construct visit the Landmine Detection module characterisation on our wiki page!
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 128
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 733
Illegal BsaI.rc site found at 922