Difference between revisions of "Team:Birkbeck/Discussion"

Revision as of 13:12, 16 September 2015

Discussion

Under construction

Sean Ross Craig (data analysis, cloning, restriction diagnostics, measurements & uploading content to the wiki), Elliott Parris (measurements & restriction diagnostics), Rachel Wellman (restriction diagnostics & measurements) & Ariana Mirzarafie-Ahi (cloning).

With thanks to Dr. Vitor Pinheiro, Dr. Jane Nicklin, Bilkis Kazi, Barbara "Babz" Steijl, Luba "Aunty" Prout.

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-<h1>Discussion</h1>
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-<h2>Under Construction</h2>
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+<h2><u><b>Discussion</b></u></h2>
+<h2>Under construction</h2>
+ <!--<h3><b><u>Signal Detection</u></b></h3>
+<p><b><u></u></b></p>
+<p>30-50 cfu/mL of sputum was the sensitivity. When targeting the 16s rRNA of Mycobacterium, the detection limit was below 30 cfu/mL of sputum (Drouillon <i>et al</i>., 2009 [they did not show this data]). This is clearly a low detection limit. Considering the components of sputum & localisation of cells, it is unclear the efficiency of transduction our product will have on the samples. <a href="https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/TBPrepKit.htm">Kits</a> are available for decontamination of sputum samples and degrading components of the sputum. Adding this step prior to exposure of the recombinant phage to samples may be a means of improving transduction efficiency and therefore reducing the detection limit.</p>
+<p>Considering our results, at time 0 minutes, there is a significant signal for P1-<i>gfp</i> expression device compared to the <i>E. coli</i> DH5α (P=0.005, with reference to <b>Fig. 9</b> in results section). This result cannot be regarded as this is probably carry over fluorescence from sub-culturing.  Photo-bleaching prior to the first reading may yield more accurate results when considering the expression of <i>gfp</i> from promoters. Also the error bars in each of the expression devices do appear excessive. A means of reducing this may be to sub-culture the cells & grow to an OD<sub>600</sub> of 0.2 and sub-culturing into experimental cultures in order to minimise the error bars.</p>
+<p>Considering culture time at 20 minutes, there is a significant signal for P1-<i>gfp</i> (P=0.002) with no difference between the OD<sub>600</sub> growth curves at this point (P=1). This is significantly less than the hours taken for results to be generated by TCR-2 (Drouillon <i>et al</i>., 2009). Cells cannot be quantified at the 20 minutes time point as no viable count was carried out, therefore the detection limit of the <i>E. coli</i> DH5α with regards to fluorescence cannot be determined.</p>
+<p>Considering our results, at 1 hour of culturing <i>E. coli</i> DH5α, there is a significant signal observed from the culture expressing the P1-<i>gfp</i> expression device (P<0.001). Considering the growth, there was no significant difference between the OD<sub>600</sub> of the P1-gfp expression device & the E. coli without plasmid (P=1 for both 50 mL cultures & 200 μL, with reference to <b>Fig. 1</b> & <b>Fig. 5</b> respectively). At the 1 hour time point, there is a significant difference between the 50 mL & 200 μL cultures (P=0.017) with the 200 μL cultures showing a higher OD<sub>600</sub>. This entails that the fluorescence of the 200 μL cultures at 1 hour corresponds to a higher culture density than 2.57×10<sup>6</sup> cfu/mL. Considering the TCR-2 method of detection, 2.57×10<sup>6</sup> cfu/mL is an increase in detection limit by a magnitude of 10^5 (30 cfu of Mycobacterium/mL of sputum) which is clearly an undesirable due to the slow growth of M. tuberculosis.</p>
+<p>As transduction was not characterised in this study, it is difficult to assess the potential limitations of this product might be.</p>-->
+ <!--The sensitivity for positive sputum samples was 88.2%, 50.4% & 80% for TRC-2, acid fast smearing & culturing respectively. (Drouillon <i>et al</i>., 2009)-->
 <br><hr><br>
 <p><b>Sean Ross Craig</b> (data analysis, cloning, restriction diagnostics, measurements & uploading content to the wiki), <b>Elliott Parris</b> (measurements & restriction diagnostics), <b>Rachel Wellman</b> (restriction diagnostics & measurements) & <b>Ariana Mirzarafie-Ahi</b> (cloning).</p>
 <br>
 <p>With thanks to <b>Dr. Vitor Pinheiro</b>, <b>Dr. Jane Nicklin</b>, <b>Bilkis Kazi</b>, <b>Barbara "<i>Babz</i>" Steijl</b>, <b>Luba "<i>Aunty</i>" Prout</b>.</p>
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