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<p>The experiment to test the tolerance was performed as follows: B. subtilis culture was divided in four treatments, varying the pH of the culture in each one; 7 (normal growth condition as a control), 8, 9, and 10. NaOH was used to induce the different changes in the pH for each treatment, and was added after one hour of cultivation. The experiment was carried out in a plate reader to follow the change of the optical density (OD 600) which correlates with bacterial growth. </p> | <p>The experiment to test the tolerance was performed as follows: B. subtilis culture was divided in four treatments, varying the pH of the culture in each one; 7 (normal growth condition as a control), 8, 9, and 10. NaOH was used to induce the different changes in the pH for each treatment, and was added after one hour of cultivation. The experiment was carried out in a plate reader to follow the change of the optical density (OD 600) which correlates with bacterial growth. </p> | ||
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<img src="https://static.igem.org/mediawiki/2017/e/e3/T--TU_Dresden--bacillusinpH.png" | <img src="https://static.igem.org/mediawiki/2017/e/e3/T--TU_Dresden--bacillusinpH.png" | ||
alt="Comparison of B.subtilis cell growth at different pH values" | alt="Comparison of B.subtilis cell growth at different pH values" | ||
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<p>For the first experiments of encapsulation, we used the B. subtilis strains TMB4131 W168 lacA::erm Pveg_sfGFP and TMB3090 W168 sacA::cat Pveg_luxABCDE. The first strain has the characteristic of expressing sfGFP in a constitutive way, which was useful to prove the presence of bacteria in the peptidosomes by detecting the fluorescence expressed by the cells. TMB3090 expresses luciferase in a constitutive way, which makes a detection of a luminescence signal possible. | <p>For the first experiments of encapsulation, we used the B. subtilis strains TMB4131 W168 lacA::erm Pveg_sfGFP and TMB3090 W168 sacA::cat Pveg_luxABCDE. The first strain has the characteristic of expressing sfGFP in a constitutive way, which was useful to prove the presence of bacteria in the peptidosomes by detecting the fluorescence expressed by the cells. TMB3090 expresses luciferase in a constitutive way, which makes a detection of a luminescence signal possible. | ||
We performed a plate reader assay using the well-scan-mode. In this mode, the whole well is scanned to detect the exact position of a signal, either fluorescence or luminescence. Its absence is displayed in a map with a green colour, while the position of the fluorescence/luminescence source appears as red. </p> | We performed a plate reader assay using the well-scan-mode. In this mode, the whole well is scanned to detect the exact position of a signal, either fluorescence or luminescence. Its absence is displayed in a map with a green colour, while the position of the fluorescence/luminescence source appears as red. </p> | ||
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+ | <figure class="makeresponsive" style="width: 70%; display: flex; align-items: center; justify-content: center; flex-direction: column;"> | ||
<img src="https://static.igem.org/mediawiki/2017/8/8a/T--TU_Dresden--platereader.png" | <img src="https://static.igem.org/mediawiki/2017/8/8a/T--TU_Dresden--platereader.png" | ||
alt="Plate reader assay, well scan mode" | alt="Plate reader assay, well scan mode" | ||
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<figcaption><b>Figure 9: Overview of well-scans of the Plate Reader Assay e </b> The results of the well-scan measurements for the detection of fluorescence and luminescence are shown. If no signal is detected, the field of the matrix is green, otherwise red.</figcaption> | <figcaption><b>Figure 9: Overview of well-scans of the Plate Reader Assay e </b> The results of the well-scan measurements for the detection of fluorescence and luminescence are shown. If no signal is detected, the field of the matrix is green, otherwise red.</figcaption> | ||
</figure> | </figure> | ||
+ | |||
<p>The well scan maps appear completely green where fluorescence/luminescence is absent, i.e. water and empty peptidosome. Wells containing a sample of the day culture and lyophilized eGFP solved in water show a red colour in the whole map. However, a localized red spot over a green background is observed where the source of fluorescence/luminescence is contained: the cells trapped inside the peptidosomes. This shows that when the peptidosome with cells inside is transferred to water, bacteria cannot diffuse away, but its kept contained within the structure, resulting in a localized red signal. </p> | <p>The well scan maps appear completely green where fluorescence/luminescence is absent, i.e. water and empty peptidosome. Wells containing a sample of the day culture and lyophilized eGFP solved in water show a red colour in the whole map. However, a localized red spot over a green background is observed where the source of fluorescence/luminescence is contained: the cells trapped inside the peptidosomes. This shows that when the peptidosome with cells inside is transferred to water, bacteria cannot diffuse away, but its kept contained within the structure, resulting in a localized red signal. </p> | ||
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<p>Nevertheless, the surface of DynaBeads can be decorated with His-Tag protein. For the “proof of principle” experiments His-Tag GFP were selected due to its availability and easy imaging procedure. First, the Invitrogen Protocol was optimized for our goals and tested for DynaBeads decoration with histidine-tagged GFP (figure X). </p> | <p>Nevertheless, the surface of DynaBeads can be decorated with His-Tag protein. For the “proof of principle” experiments His-Tag GFP were selected due to its availability and easy imaging procedure. First, the Invitrogen Protocol was optimized for our goals and tested for DynaBeads decoration with histidine-tagged GFP (figure X). </p> | ||
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<img src="https://static.igem.org/mediawiki/2017/8/84/T--TU_Dresden--Dynabeads.png" | <img src="https://static.igem.org/mediawiki/2017/8/84/T--TU_Dresden--Dynabeads.png" | ||
alt="Dynabeads labeled with histidine-tagged GFP in the Binding/Wash buffer." | alt="Dynabeads labeled with histidine-tagged GFP in the Binding/Wash buffer." | ||
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<p>To prove the surface of the Dynabeads enveloped to the Peptidosome membrane availability for the decoration the labeling procedure was applied after the Peptidosome formation in both Binding/Wash Buffer and LB media (Picture 2). </p> | <p>To prove the surface of the Dynabeads enveloped to the Peptidosome membrane availability for the decoration the labeling procedure was applied after the Peptidosome formation in both Binding/Wash Buffer and LB media (Picture 2). </p> | ||
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<img src="https://static.igem.org/mediawiki/2017/2/26/T--TU_Dresden--peptidosomegfp.png" | <img src="https://static.igem.org/mediawiki/2017/2/26/T--TU_Dresden--peptidosomegfp.png" | ||
alt="Peptidosome with Dynabeads labeled with His-Tag GFP in Binding/Wash Buffer (WB) or LB media (LB)" | alt="Peptidosome with Dynabeads labeled with His-Tag GFP in Binding/Wash Buffer (WB) or LB media (LB)" |
Revision as of 23:29, 30 October 2017