Team:ULB-Brussels/Project/Results

From 2014.igem.org

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<h2>Activity of the alkaline phosphatase with a proline on its N-terminal extremity</h2>
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<h1>Activity of the alkaline phosphatase with a proline on its N-terminal extremity</h1>
In order to test the functionality of the alcaline phosphatase (phoA) with a proline (P) on its N-terminal extremity, we constructed by restriction processes different plasmids:  
In order to test the functionality of the alcaline phosphatase (phoA) with a proline (P) on its N-terminal extremity, we constructed by restriction processes different plasmids:  
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<h2>Characterization of our biobrick ccdB</h2>
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<h1>Characterization of our biobrick ccdB</h1>
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<p>See the <a href="http://parts.igem.org/Part:BBa_K1318000><b>registry page</b></a></p>
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In order to characterize the ccdB biobrick, we sent the biobricks to sequencing and made a screen of activity for the protein ccdB.</p>
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Here is the link to our part registry where information can be found about it
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$\rightarrow$ <a href="http://parts.igem.org/Part:BBa_K1318000"> ULB-Brussels part <b>(registry page)</b></a> </p>
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As a screen we did a killing assay, because of the toxic property of ccdB.</p>
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We constructed 4 different colonies including a control colony made of E.coli without plasmid (line 1), a second one with pBAD33::ccdB (line2), a third one containing pKK233::ccdA (line 3) and the final one with both plasmids.</p>
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The ccdA gene encoded for a protein wich acts as an anti-toxin for ccdB and so allows the bacteria which express it to survive.</p>
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The two following screens show us the results of the killing assay on two different media. To interpret them one should know that IPTG induces pKK233’s expression, glucose represses pBAD33’s expression and arabinose incude the expression of pBAD33.</p>We made dilution to assure that the cell concentration didn’t affect the toxicity or anti-toxicity. <!--The columns from left to right: $10^{0}$, $10^{-2}$, $10^{-3}$, $10^{-4}$, $10^{-6}$.-->
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<br>
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<center> A. <img src="https://static.igem.org/mediawiki/2014/d/dd/Test-ccd-iGEM-2-%2B.png">
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B. <img src="https://static.igem.org/mediawiki/2014/d/dd/Test-ccd-iGEM-2-%2B.png"></center>
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<br><font size="1"><b>Figure 6 </b>$:\hspace{0.16cm}$
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CcdB Killing assay on the bacterial strain containing a plasmid pKK233/ccdA inductible with IPTG and/or a plasmid pBAD33/ccdB inductible with arabinose according various dilutions.
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A. Under glucose & IPTG condition. B. Under arabinose & IPTG condition.</font>
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<!-- Ex-imgs:
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$\small IPTG$ $\hspace{0.2cm}+\hspace{0.2cm} glucose:\hspace{2cm}$
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<img src="https://static.igem.org/mediawiki/2014/d/dd/Test-ccd-iGEM-2-%2B.png"></p>
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$\small IPTG$ $\hspace{0.2cm}+\hspace{0.2cm} arabinose:\hspace{1.65cm}$
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<img src="https://static.igem.org/mediawiki/2014/1/11/Test-ccd-iGEM-3-%2B.png"></p>
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<img src="https://static.igem.org/mediawiki/2014/a/a8/Test-ccd-iGEM-1-%2B.png"></p>
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<p>On  the first media containing IPTG and glucose: each colony grew. </p>
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On the media containing IPTG and arabinose the strand with pBAD33::ccdB is killed and the strand with both ccdA and ccdB grew as well as the other two colonies.</p>
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According to the results shown on the first media we have been assured that ccdA is non-toxic for the bacteria and would not be responsable for their death on the next experience, we have also seen that glucose does repress the expression of the ccdB gene.</p>
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The second screen allowed us to say that while it is expressed ccdB is toxic for the bacteria and leads to their death whereas the expressions of both ccdB and its anti-toxine ccdA enable the bacteria to survive.</p>
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In conclusion, the screen of the activity of ccdB has been a success. We have shown that ccdB is active as a toxin wich kills bacteria and that the anti-toxine ccdA inhibts its toxicity allowing bacteria with the two genes expressed to survive.</p>
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Revision as of 15:22, 11 October 2014

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- Université Libre de Bruxelles -


Results


Retrieved from "http://2014.igem.org/Team:ULB-Brussels/Project/Results"

Activity of the alkaline phosphatase with a proline on its N-terminal extremity

In order to test the functionality of the alcaline phosphatase (phoA) with a proline (P) on its N-terminal extremity, we constructed by restriction processes different plasmids: pBAD33:: phoA+P (on top), pBAD33:: phoA (in the middle) and we used pBAD33 as a control (in the bottom). Then we chemoporated the plasmids into a lacking phoA strain and spread the bacteria on different chromogenic culture media including media with glucose (A.2), with arabinose (B.2), and without glucose nor arabinose (A.1 $\small\&$ B.1) as shown in the figure below.



Figure 5 $:\hspace{0.16cm}$ A. Comparison of the phosphatase activity of a strain DELTA-pho with a pBAD33::phoA::proline construction (top), a pBAD33::phoA construction (middle) and an empty pBAD33 control (bottom) under neither glucose nor arabinose condition (A.1) & under glucose repression (A.2). $\hspace{0.16cm}$ B. Same comparison, under neither glucose nor arabinose condition (B.1) $\scriptsize\&$ under arabinose induction (B.2).

The expression of the plasmid is induced by arabinose and repressed by glucose and the chromogenic media enable the bacteria whose alkaline phosphatase is active to color in blue.

We have observed that each colony has been colored in a light blue when the plasmid was under the repression of glucose (A.2) while only the bacteria which have phoA or phoA+P within their plasmid appeared in a strong blue at the arabinose media (B.2). The same result was observed at the media without arabinose nor glucose but the blue was lighter.

First, we think, following the results under glucose repression, that the production of something (that we didn’t characterize) has been induced by glucose in the bacteria because each strain shows the same degree of blue.

Secondly, at the media without arabinose or glucose, we did not observe a blue color appear in the control colony but we still observed it in the other two colonies. We think this is probably due to a small expression of the genes phoA and phoA+P.

Finally looking at the arabinose media, we observed a strong blue color in the colonies with the phoA and phoA+P genes while the control colony did not color itself in blue.

In conclusion, a proline on the N-terminal extremity of the alkaline phosphatase does not inhibit its activity.



Characterization of our biobrick ccdB

See the

< Notebook