Difference between revisions of "Part:BBa K1316003"

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	<h3>Characterisation</h3>		<h3>Characterisation</h3>
	<p>		<p>
−	Different type of experiments were conducted to characterize this BioBrick:	+	Different type of experiments were conducted to characterize this BioBrick.
		+	</p>
		+	<br>
		+
		+	<p>
		+	The different constructs used are:
		+	<ul>
		+	<li> p[F]::mKATE, also referred here as LD2  </li>
		+	<li> p[J]::mKATE, also referred here as LD3  </li>
		+	<li> p[F] incl. N-enzymes, also referred here  as LD4  </li>
		+	<li> p[J] incl. N-enzymes, also referred here as LD5  </li>
		+	<li> p[F]::mKATE p[J]::mKATE, also referred here  as LD6  </li>
		+	</ul>
		+
	</p>		</p>
	<br>		<br>

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Revision as of 11:51, 16 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.

The different constructs used are:

• p[F]::mKATE, also referred here as LD2
• p[J]::mKATE, also referred here as LD3
• p[F] incl. N-enzymes, also referred here as LD4
• p[J] incl. N-enzymes, also referred here as LD5
• p[F]::mKATE p[J]::mKATE, also referred here as LD6

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.

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 2,4-DNT.

For more information about the characterisation of this construct visit the Landmine Detection module characterisation on our wiki page!

Sequence and Features