Difference between revisions of "Part:BBa K1316008"

Latest revision as of 21:41, 17 October 2014

ybiJ promoter coupled to mKate2 reporter gene and Rhamnose promoter coupled to N-genes

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.

nfsA, nfsB and nemA (refered here as N-genes) are the genes for NfsA, NfsB and NEM reductases, which presumably play a role in 2,4-DNT and 2,4,6-TNT metabolism because when these genes were knocked out, the response of the yqjF and ybiJ promoters decreased 1,000 times.[1] These genes are regulated by the inducible Rhamnose promoter 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. The presence of the N-genes aims to enhance this response.

Characterisation

Different type of experiments were conducted to characterize the Landmine Detection BioBricks.

The different constructs used are:

• p[F]::mKATE, also referred here as LD2, which corresponds to BioBrick BBa_K1316003
• p[J]::mKATE, also referred here as LD3, which corresponds to BioBrick BBa_K1316005
• p[F] incl. N-enzymes, also referred here as LD4, which corresponds to BioBrick BBa_K1316007
• p[J] incl. N-enzymes, also referred here as LD5, which corresponds to BioBrick BBa_K1316008
• p[F]::mKATE p[J]::mKATE, also referred here as LD6, which corresponds to BioBrick BBa_K1316009

Plate Reader

A plate reader is a machine designed to handle samples on 6-1536 well format microtiter plates for the measuring of physical properties such as absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarisation. Concerning this module, the plate reader device was used for the measurement of fluorescence intensity generated by cells carrying the BioBricks designed to detect land mines. The final protocol developed for Plate reader analysis for this module can be found by clicking on this link.

Results - Plate Reader

Using the different final Landmine detection constructs LD2-6, different concentrations of 2,4-DNT were tested (figure 1). 2,4-DNT, due to safety reasons, was bought in liquid solution dissolved in acetonitrile. To test that the compound triggering the response of the promoters is 2,4-DNT and not acetonitrile, the 0 mg/L 2,4-DNT solution was prepared with the same concentration of acetonitrile contained in the most concentrated 2,4-DNT solution.

The fact that the positive control (constitutively expressed mKate2) presents a clear fluorescence signal at 0 and 50 mg/L, but not at 100 mg/L indicates that high concentrations of 2,4-DNT are toxic for cells after several hours. The toxic compound seems to be 2,4-DNT and not acetonitrile because the sample at 0 mg/L 2,4-DNT (same acetonitrile concentration as the 100 mg/L 2,4-DNT sample) did not kill the cells. Constructs LD2, LD3 and LD6 as well as LD4 and LD5 with no induction of the N-genes show no clear mKate2 induction over time. Not many conclusions can be drawn form LD2 due to the big standard deviation. However, when the N-genes are induced with L-Rhamnose, the constructs LD4 and LD5 showed a clear increase in fluorescence over time for a concentration of 50 mg/L 2,4-DNT. In this situation, besides, LD5 seems to be more sensitive, as there is less leakage (there is less fluorescence signal at 0 mg/L DNT).

It could still be that at 50 mg/L 2,4-DNT, what triggers the response of the promoters is actually the acetonitrile and not the 2,4-DNT, but that would be strange because at 0 mg/L 2,4-DNT (acetonitrile is 2 times more concentrated than at 50 mg/L 2,4-DNT) the cells survive and there is no fluorescence response.

This data suggests that both promoters, ybiJ and yqjF, respond to 2,4-DNT; and that the presence of the N-genes is of high importance for this response. Hence, we conclude that the two best BioBricks for Landmine detection are (in this order) LD5 (p[J] incl. N-enzymes) and LD4 (p[F] incl. N-enzymes), and in order to obtain a good result the N-genes must be expressed.

References

[1] S. Yagur-Kroll, S. Belkin et al., “Escherichia Coli bioreporters for the detection of 2,4-dinitrotoluene and 2,4,6-trinitrotoluene”, Appl. Microbiol. Biotechnol. 98, 885-895, 2014.