Team:LMU-Munich/Data/Sporepurification

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Latest revision as of 13:37, 26 October 2012

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Sporobead purification

To be able to use our Sporobeads, we have to make ensure that all remaining vegetative cells in the culture need to be thoroughly removed. In order to purify our spores, we treated culture samples that were grown for 24 hours in Difco Sporulation Medium (see protocol for enhancement of mature spore numbers) with three different methods: French Press, sonification and lysozyme. The results are shown in the following table.

	all cells	mature spores	immature spores

untreated wildtype	7.29 x 10⁸ /ml	1 x 10⁸ /ml 13.71%	0.04 x 10⁸ /ml 0.55%
untreated P_cotYZ-cotZre-gfp-terminator	6.79 x 10⁸ /ml	1 x 10⁸ /ml 14.72%	0.13 x 10⁸ /ml 1.9%
French Press wildtype	4.87 x 10⁸ /ml	2.1 x 10⁸ /ml 43%	0.05 x 10⁸ /ml 1%
French Press P_cotYZ-cotZre-gfp-terminator	4.75 x 10⁸ /ml	1.88 x 10⁸ /ml 39.58%	0.05 x 10⁸ /ml 1%
Sonification wildtype	4.6 x 10⁸ /ml	1.22 x 10⁸ /ml 26.52%	0.1 x 10⁸ /ml 2%
Sonification P_cotYZ-cotZre-gfp-terminator	6.72 x 10⁸ /ml	1.53 x 10⁸ /ml 22.77%	0.23 x 10⁸ /ml 3%
Lysozyme wildtype	2.48 x 10⁸ /ml	1.58 x 10⁸ /ml 63.7%	0 /ml
Lysozyme P_cotYZ-cotZre-gfp-terminator	1.05 x 10⁸ /ml	1.05 x 10⁸/ml 100%	0 /ml

The data demonstrates that after treatment with French Press and ultrasound (sonification) the number of spores compared to the untreated samples were increased. We assume this to be an experimental artifact, since it was not always possible to distinguish between mature spores and cell debris during counting. However, a huge difference between the number of vegetative cells and spores was observed for lysozyme-treated samples as visualized in the pictures below (Fig. 1). Because the lysed vegetative cells, it was easy to recognize the mature spores.

Fig. 1: Phase contrast pictures after different treatments

Next, we tested the fluorescence of Sporobeads after lysozyme treatment. This analysis revealed that lysozyme did not harm the gfp-fusion proteins, since fluorescence was not altered (see Fig. 2).

Fig. 2: Fluorescence of wild type spores and Sporobeads after treatment with lysozyme. Sporobeads still show undeminished fluorescence activity.

Retrieved from "http://2012.igem.org/Team:LMU-Munich/Data/Sporepurification"

@@ Line 10: / Line 10: @@
-<p align="justify">To purify our spores from the vegetative cells we treated the samples, that were grown for 24 hours in DS-Medium, with three different methods. The data is summarized in the following table:</p>
+<p align="justify">To be able to use our '''Sporo'''beads, we have to make ensure that all remaining vegetative cells in the culture need to be thoroughly removed. In order to purify our spores, we treated culture samples that were grown for 24 hours in Difco Sporulation Medium (see [https://static.igem.org/mediawiki/2012/e/e9/LMU-Munich_2012_Protocol_for_enhancement_of_mature_spore_numbers.pdf protocol for enhancement of mature spore	numbers]) with three different methods: French Press, sonification and lysozyme. The results are shown in the following table.</p>
@@ Line 30: / Line 30: @@
 |0.04 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 0.55% </font>
 |-
-!untreated <font color="#EBFCE4" size="2">P<sub>''cotYZ''</sub>-''cotZre''-''gfp''-''terminator''</font>
+!untreated <font color="#EBFCE4" size="2">P<sub>''cotYZ''</sub>-''cotZre''-''gfp''-terminator</font>
 |6.79 ''x'' 10<sup>8</sup> /ml
 |1 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 14.72% </font>
@@ Line 40: / Line 40: @@
 |0.05 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 1% </font>
 |-
-!French Press <font color="#EBFCE4" size="2">P<sub>''cotYZ''</sub>-''cotZre''-''gfp''-''terminator''</font>
+!French Press <font color="#EBFCE4" size="2">P<sub>''cotYZ''</sub>-''cotZre''-''gfp''-terminator</font>
 |4.75 ''x'' 10<sup>8</sup> /ml
 |1.88 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 39.58% </font>
@@ Line 50: / Line 50: @@
 |0.1 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 2% </font>
 |-
-!Sonification <font color="#EBFCE4" size="2">P<sub>''cotYZ''</sub>-''cotZre''-''gfp''-''terminator''</font>
+!Sonification <font color="#EBFCE4" size="2">P<sub>''cotYZ''</sub>-''cotZre''-''gfp''-terminator</font>
 |6.72 ''x'' 10<sup>8</sup> /ml
 |1.53 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 22.77% </font>
@@ Line 60: / Line 60: @@
 |0 /ml
 |-
-!Lysozyme <font color="#EBFCE4" size="2">P<sub>''cotYZ''</sub>-''cotZre''-''gfp''-''terminator''</font>
+!Lysozyme <font color="#EBFCE4" size="2">P<sub>''cotYZ''</sub>-''cotZre''-''gfp''-terminator</font>
 |1.05 ''x'' 10<sup>8</sup> /ml
 |1.05 ''x'' 10<sup>8</sup>/ml <br><font size="2"> 100% </font>
@@ Line 68: / Line 68: @@
-<p align="justify">The table shows that after treatment with French Press and ultrasound the number of spores compared to the untreated samples was increased. We assume the reason for this was the impurity of these samples that derived from damaged vegetative cells. Thus, during counting it was not always possible to distinguish between mature spores and cell waste. However, a huge difference in number of vegetative cells and spores between untreated and lysozyme treated samples was noticeable under microscopy as it is visualized in the table above and the pictures below. Because the vegetative cells were lyzed and not damaged it was easy to recognize the mature spores.
+<p align="justify">The data demonstrates that after treatment with French Press and ultrasound (sonification) the number of spores compared to the untreated samples were increased. We assume this to be an experimental artifact, since it was not always possible to distinguish between mature spores and cell debris during counting. However, a huge difference between the number of vegetative cells and spores was observed for lysozyme-treated samples as visualized in the pictures below (Fig. 1). Because the lysed vegetative cells, it was easy to recognize the mature spores.
 </p>
+{| style="color:black;" cellpadding="3" width="100%" cellspacing="0" border="0" align="center" style="text-align:left;"
+| style="width: 100%;background-color: #EBFCE4;" |
+{|
+|[[File:Treatments.png|610px|center]]
+|-
+| style="width: 70%;background-color: #EBFCE4;" |
+{| style="color:black;" cellpadding="0" width="70%" cellspacing="0" border="0" align="center" style="text-align:center;"
+|style="width: 70%;background-color: #EBFCE4;" |
+<font color="#000000"; size="2">Fig. 1: Phase contrast pictures after different treatments</font>
+|}
+|}
+|}
+<p align="justify">Next, we tested the fluorescence of '''Sporo'''beads after lysozyme treatment. This analysis revealed that lysozyme did not harm the gfp-fusion proteins, since fluorescence was not altered (see Fig. 2).</p>
+{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"
+| style="width: 100%;background-color: #EBFCE4;" |
+{|
+|[[File:Fluorescence after lysozyme.png|400px|center]]
+|-
+| style="width: 70%;background-color: #EBFCE4;" |
+{| style="color:black;" cellpadding="0" width="90%" cellspacing="0" border="0" align="center" style="text-align:center;"
+|style="width: 70%;background-color: #EBFCE4;" |
+<font color="#000000"; size="2"><p align="justify">Fig. 2: Fluorescence of wild type spores and '''Sporo'''beads after treatment with lysozyme. '''Sporo'''beads still show undeminished fluorescence activity. </p></font>
+|}
+|}
+|}
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