Part:BBa_K1316008
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
Using the different final Landmine detection constructs LD2-6, different concentrations of 2,4-DNT were tested (figure 1). 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 DNT has the same acetonitrile concentration as the 100 mg/L DNT sample. 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, 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).
From this data we concluded 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 to obtain a good result the N-genes must be expressed.
For more information about the characterisation of this construct visit the Landmine Detection module characterisation on our wiki page!
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 381
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 886
Illegal BsaI.rc site found at 1075
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