Team:CityU HK/project/result fad

From 2014.igem.org

(Difference between revisions)
 
Line 84: Line 84:
<p class="content">Quantitative real-time PCR analyses showed that <i>fadL</i> and <i>fadD</i> mRNAs were increased in recombinant <i>fadL-fadD E. coli</i> as compared to the control cells in the absence and presence of IPTG induction  (Figures 1A and 1B). For the fatty acid uptake analysis, oleic acid concentration in recombinant and controls cells was measured by GC-MS. Although no difference in oleic acid uptake was observed in the <i>fadL-fadD</i> recombinant and control cells at 0 µM and 40 µM of IPTG, the oleic acid concentration in the <i>fadL-fadD</i> transformant was 2-fold higher at 400 µM IPTG than the control cells (Figure 2). Our overall results, show no apparent correlation in <i>fadL</i> and <i>fadD</i> mRNA expression levels and oleic acid uptake in <i>E. coli</i>. However, due to time constraint, these experiments were performed only once, and further replicates will need to be carried out in future studies in order to determine the statistical significance of the data.</p><br>
<p class="content">Quantitative real-time PCR analyses showed that <i>fadL</i> and <i>fadD</i> mRNAs were increased in recombinant <i>fadL-fadD E. coli</i> as compared to the control cells in the absence and presence of IPTG induction  (Figures 1A and 1B). For the fatty acid uptake analysis, oleic acid concentration in recombinant and controls cells was measured by GC-MS. Although no difference in oleic acid uptake was observed in the <i>fadL-fadD</i> recombinant and control cells at 0 µM and 40 µM of IPTG, the oleic acid concentration in the <i>fadL-fadD</i> transformant was 2-fold higher at 400 µM IPTG than the control cells (Figure 2). Our overall results, show no apparent correlation in <i>fadL</i> and <i>fadD</i> mRNA expression levels and oleic acid uptake in <i>E. coli</i>. However, due to time constraint, these experiments were performed only once, and further replicates will need to be carried out in future studies in order to determine the statistical significance of the data.</p><br>
-
<img class="displayed" src="https://static.igem.org/mediawiki/2014/0/0e/CityU_HK_project_result_fad1.png" width="35%"><br>
+
<img class="displayed" src="https://static.igem.org/mediawiki/2014/0/0e/CityU_HK_project_result_fad1.png" width="40%"><br>
-
<img class="displayed" src="https://static.igem.org/mediawiki/2014/c/c8/CityU_HK_project_result_fad2.png" width="35%"><br>
+
<img class="displayed" src="https://static.igem.org/mediawiki/2014/c/c8/CityU_HK_project_result_fad2.png" width="40%"><br>
<table>
<table>
Line 95: Line 95:
</table>
</table>
-
<br><img class="displayed" src="https://static.igem.org/mediawiki/2014/4/45/CityU_HK_project_result_fad3.png" width="35%"><br>
+
<br><img class="displayed" src="https://static.igem.org/mediawiki/2014/4/45/CityU_HK_project_result_fad3.png" width="40%"><br>
<table>
<table>

Latest revision as of 07:08, 17 October 2014

Bootstrap 101 Template

Enhancement of Fatty acid Uptake by Overexpression of FadL and FadD in Escherichia coli.

Overview :

In an attempt to increase fatty acid uptake in E. coli, we constructed a tetracycline-repressible fadL-fadD expression system (BBa_K142606). As we were aiming to ensure significant co-expression of the fadL-fadD genes in E.coli, we subcloned the fadL-fadD gene cassette to the IPTG-inducible pSNAP tag (T7)-2 expression vector to test the effect of FadL-FadD overexpression on oleic acid uptake. Expression of the fadL and fadD genes in recombinant and control E. coli host cells was measured by qRT-PCR assays, and fatty acid uptake measured by GC-MS analysis.



Results :

Quantitative real-time PCR analyses showed that fadL and fadD mRNAs were increased in recombinant fadL-fadD E. coli as compared to the control cells in the absence and presence of IPTG induction (Figures 1A and 1B). For the fatty acid uptake analysis, oleic acid concentration in recombinant and controls cells was measured by GC-MS. Although no difference in oleic acid uptake was observed in the fadL-fadD recombinant and control cells at 0 µM and 40 µM of IPTG, the oleic acid concentration in the fadL-fadD transformant was 2-fold higher at 400 µM IPTG than the control cells (Figure 2). Our overall results, show no apparent correlation in fadL and fadD mRNA expression levels and oleic acid uptake in E. coli. However, due to time constraint, these experiments were performed only once, and further replicates will need to be carried out in future studies in order to determine the statistical significance of the data.




Figure 1. Quantitative RT-PCR analysis of fadL and fadD mRNAs upon induction with IPTG a various concentrations. (A). Expression of fadL in control E. coli BL21(DE3) host cells (BLUE) and fadL-fadD transformant (RED). (B). Expression of fadD in control E. coli BL21(DE3) host cells (BLUE) and fadL-fadD transformant (RED). E. coli cells were cultured in LB + oleic acid (3.5 µM) medium and induced with 0 µM, 40 µM or 400 µM IPTG for 6 hrs. Cells were harvested for total RNA extraction and lipid extraction for GC-MS analyses.


Figure 2. Oleic acid concentration in fadL-fadD transformant and control E. coli cells at various concentrations of IPTG. Intracellular concentration of oleic acid in control E. coli BL21(DE3) host cells (YELLOW) and fadL-fadD transformant (GRAY). E. coli cells were incubated in LB + oleic acid (10 µM) medium and induced with 0 µM, 40 µM or 400 µM IPTG for 6 hrs.


Bootstrap 101 Template