Team:TU Delft-Leiden/WetLab/landmine/characterisation

From 2014.igem.org

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The different assays used to test our Land Mine BioBricks are:
The different assays used to test our Land Mine BioBricks are:
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<ul>
  <li><a href="/Team:TU_Delft-Leiden/WetLab/landmine/characterisation#LDplate_reader"><p>Plate Reader </p>
  <li><a href="/Team:TU_Delft-Leiden/WetLab/landmine/characterisation#LDplate_reader"><p>Plate Reader </p>
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         </a>
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         </a>
         </a>
</li>
</li>
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</ul>
</p>
</p>
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<p>
<p>
The different constructs used are:
The different constructs used are:
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<ul>
<li> BBa_K1316003, referred from now on as LD2  </li>
<li> BBa_K1316003, referred from now on as LD2  </li>
<li> BBa_K1316005, referred from now on as LD3  </li>
<li> BBa_K1316005, referred from now on as LD3  </li>
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<li>BBa_K1316008, referred from now on as LD5  </li>
<li>BBa_K1316008, referred from now on as LD5  </li>
<li>BBa_K1316009, referred from now on as LD6  </li>
<li>BBa_K1316009, referred from now on as LD6  </li>
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</ul>
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<p>
<p>
<figcaption>
<figcaption>
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Figure 1: Fluorescent signal measured on the plate reader.
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Figure 1: Fluorescence signal measured on the plate reader.
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<p>
<p>
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The BioBricks showed an increasing fluorescent signal over time when they were induced with DNT. When non-induced (0mg/L DNT), the constructs showed no clear increase in fluorescent signal.
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The BioBricks showed an increasing fluorescence signal over time when they were induced with DNT. When non-induced (0mg/L DNT), the constructs showed no clear increase in fluorescence signal.
The construct LD2 (sample B on the Figures) showed a much higher response than LD3 (sample C on the Figures), hence, the yqjF promoter responds better to DNT than the ybiJ promoter, consistently with the literature [1].
The construct LD2 (sample B on the Figures) showed a much higher response than LD3 (sample C on the Figures), hence, the yqjF promoter responds better to DNT than the ybiJ promoter, consistently with the literature [1].
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The non-induction of the negative control (sample D on the Figures) indicates that it is the presence of the promoter that generates the signal in front of the presence of DNT.
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The induced negative control (sample D on the Figures) shows no fluorescence, which indicates that the yqjF promoter is positively induced by 2,4-DNT.
<a name="LDmicroscopy"></a>  
<a name="LDmicroscopy"></a>  
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<h4> FACS </h4>
<h4> FACS </h4>
<p>
<p>
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Fluorescence-activated cell sorting (FACS) is a specialised type of flow cytometry that allows the separation of individual cells based on the specific light scattering and fluorescent characteristics of each cell. Using FACS, information can be known of the size, shape and fluorescence of individual cells, therefore, it is a technique that can be used to observe the fluorescent response of our Landminde detection BioBricks in front of DNT.
+
Fluorescence-activated cell sorting (FACS) is a specialised type of flow cytometry that allows the separation of individual cells based on the specific light scattering and fluorescent characteristics of each cell. Using FACS, information can be attained of the size, shape and fluorescence of individual cells, therefore, it is a technique that can be used to observe the fluorescent response of our Landminde detection BioBricks in front of DNT.
</p>
</p>
<br>
<br>
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The FACS technology allows us to see that, per cell, more fluorescence is produced by the construct LD2 after several hours of their induction with DNT (figure 2 bottom) compared to the early stages of induction (figure 2 top). Figure 3 clearly shows the increase in fluorescent signal of the two cultures carrying the LD2 BioBrick.
+
The FACS technology allows us to see that, per cell, more fluorescence is produced by the construct LD2 after several hours of their induction with DNT (figure 2 bottom) compared to the early stages of induction (figure 2 top). Figure 3 clearly shows the increase in fluorescence signal of the two cultures carrying the LD2 BioBrick.
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<img src="https://static.igem.org/mediawiki/2014/9/98/TUDelft_2014_Few_induction.jpg" width="75%" height="75%">
<img src="https://static.igem.org/mediawiki/2014/9/98/TUDelft_2014_Few_induction.jpg" width="75%" height="75%">
<figcaption>
<figcaption>
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Figure 2: Fluorescent signal emited by cells carrying constitutively expressed mKate2 (positive control), two paralel samples of the construct LD2 (BBa_K1316003) (Samples 1 and 2), and empty cells not carrying any BioBrick (negative control) 2 hours after induction (left) and 6 hours after induction (right).
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Figure 2: Fluorescence signal emitted by cells carrying constitutively expressed mKate2 (positive control), two parallel samples of the construct LD2 (BBa_K1316003) (Samples 1 and 2), and empty cells not carrying any BioBrick (negative control) 2 hours after induction (left) and 6 hours after induction (right).
</figcaption>
</figcaption>
</figure>
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<img src="https://static.igem.org/mediawiki/2014/3/34/ITUDelft_2014_GEM2014_Page_07.jpg" width="75%" height="75%">
<img src="https://static.igem.org/mediawiki/2014/3/34/ITUDelft_2014_GEM2014_Page_07.jpg" width="75%" height="75%">
<figcaption>
<figcaption>
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Figure 3: Fluorescent signal emited by cells carrying the construct LD2 (BBa_K1316003) of two different cultures (Top and Bottom) 2 hours after DNT induction (sample 1 and 2) and 6 hours after DNT induction (samples 1 after and 2 after).
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Figure 3: Fluorescence signal emitted by cells carrying the construct LD2 (BBa_K1316003) of two different cultures (Top and Bottom) 2 hours after DNT induction (sample 1 and 2) and 6 hours after DNT induction (samples 1 after and 2 after).
</figcaption>
</figcaption>
</figure>
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<h3> Conclusions </h3>
<h3> Conclusions </h3>
From the assays performed it can be concluded that:  
From the assays performed it can be concluded that:  
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<li>BioBrick LD2 is the best performing for the detection of DNT. The other BioBricks seem to have a probably too weak response to be able to generate a biosensor out of them.  </li>
+
<li>BioBrick LD2 is the best performing for the detection of DNT. The other BioBricks seem to present a too weak response to be able to generate a biosensor out of them.  </li>
-
<li>The yqjF promoter from BioBrick LD2 is activated in front of the presence of DNT, and the FACS assay is probably the experiment that better shows it as it shows the increase of fluorescence per cell 6 hours after induction with DNT. </li>
+
<li>The yqjF promoter from BioBrick LD2 is activated in the presence of DNT, and the FACS assay is probably the experiment that better shows it as it shows the increase of fluorescence per cell 6 hours after induction with DNT. </li>

Revision as of 22:12, 15 October 2014

Module Landmine Detection - Characterization

click to return to the  Module Landmine Detection


As already mentioned, the promoters found to be activated in presence of several chemical compounds that can leak from land mines (ybiJ and yqjF) were coupled to the expression of the fluorescent protein mKate2.

Assays

The different assays used to test our Land Mine BioBricks are:


The different constructs used are:

  • BBa_K1316003, referred from now on as LD2
  • BBa_K1316005, referred from now on as LD3
  • BBa_K1316007, referred from now on as LD4
  • BBa_K1316008, referred from now on as LD5
  • BBa_K1316009, referred from now on as LD6


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 constructs LD2 and LD3, different concentrations of 2,4-DNT were tested:

Figure 1: Fluorescence signal measured on the plate reader.

The BioBricks showed an increasing fluorescence signal over time when they were induced with DNT. When non-induced (0mg/L DNT), the constructs showed no clear increase in fluorescence signal. The construct LD2 (sample B on the Figures) showed a much higher response than LD3 (sample C on the Figures), hence, the yqjF promoter responds better to DNT than the ybiJ promoter, consistently with the literature [1]. The induced negative control (sample D on the Figures) shows no fluorescence, which indicates that the yqjF promoter is positively induced by 2,4-DNT.

Microscopy

FACS

Fluorescence-activated cell sorting (FACS) is a specialised type of flow cytometry that allows the separation of individual cells based on the specific light scattering and fluorescent characteristics of each cell. Using FACS, information can be attained of the size, shape and fluorescence of individual cells, therefore, it is a technique that can be used to observe the fluorescent response of our Landminde detection BioBricks in front of DNT.


The FACS technology allows us to see that, per cell, more fluorescence is produced by the construct LD2 after several hours of their induction with DNT (figure 2 bottom) compared to the early stages of induction (figure 2 top). Figure 3 clearly shows the increase in fluorescence signal of the two cultures carrying the LD2 BioBrick.
Figure 2: Fluorescence signal emitted by cells carrying constitutively expressed mKate2 (positive control), two parallel samples of the construct LD2 (BBa_K1316003) (Samples 1 and 2), and empty cells not carrying any BioBrick (negative control) 2 hours after induction (left) and 6 hours after induction (right).
Figure 3: Fluorescence signal emitted by cells carrying the construct LD2 (BBa_K1316003) of two different cultures (Top and Bottom) 2 hours after DNT induction (sample 1 and 2) and 6 hours after DNT induction (samples 1 after and 2 after).

Conclusions

From the assays performed it can be concluded that:
  • BioBrick LD2 is the best performing for the detection of DNT. The other BioBricks seem to present a too weak response to be able to generate a biosensor out of them.
  • The yqjF promoter from BioBrick LD2 is activated in the presence of DNT, and the FACS assay is probably the experiment that better shows it as it shows the increase of fluorescence per cell 6 hours after induction with DNT.

    • 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.

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