Line 160: | Line 160: | ||
<p>Before the encapsulation of <i>B. subtilis</i> inside the peptidosomes, it was necessary to perform experiments to study possible interactions between the organism and the dipeptide, for example, whether the bacteria can use Fmoc-FF as a nitrogen source, or if the organism can survive the process of encapsulation, since the bacterial pellet is resuspended in the alkaline Fmoc-FF solution. </p> | <p>Before the encapsulation of <i>B. subtilis</i> inside the peptidosomes, it was necessary to perform experiments to study possible interactions between the organism and the dipeptide, for example, whether the bacteria can use Fmoc-FF as a nitrogen source, or if the organism can survive the process of encapsulation, since the bacterial pellet is resuspended in the alkaline Fmoc-FF solution. </p> | ||
<br> | <br> | ||
− | <h3> Can Fmoc-FF use <i>B. subtilis</i> as a nitrogen source?</h3> | + | <h3> Can Fmoc-FF use <i><b>B. subtilis</b></i> as a nitrogen source?</h3> |
<p>Special culture plates were created using Jensen’s medium. This type of medium has all the basic nutrients that bacteria needs for its development, with exception of nitrogen. </p> | <p>Special culture plates were created using Jensen’s medium. This type of medium has all the basic nutrients that bacteria needs for its development, with exception of nitrogen. </p> | ||
Line 176: | Line 176: | ||
<p>Treatment 1 was a negative control, since the strain of <i>B. subtilis</i> (wildtype) cannot develop in absence of nitrogen, so no growth at all was expected. Treatment 2 was the positive control: with the nitrogen supplement, <i>B. subtilis</i> should be able to grow. These expectations corresponded with reality: colonies grew in treatment 2 but none in treatment 1. Treatments 3 and 4 did not result in colony growth either, meaning that the Fmoc-FF cannot be used as a source of nitrogen for the bacteria. </p> | <p>Treatment 1 was a negative control, since the strain of <i>B. subtilis</i> (wildtype) cannot develop in absence of nitrogen, so no growth at all was expected. Treatment 2 was the positive control: with the nitrogen supplement, <i>B. subtilis</i> should be able to grow. These expectations corresponded with reality: colonies grew in treatment 2 but none in treatment 1. Treatments 3 and 4 did not result in colony growth either, meaning that the Fmoc-FF cannot be used as a source of nitrogen for the bacteria. </p> | ||
<br> | <br> | ||
− | <h3> Tolerance of <i>B. subtilis</i> against alkaline pH</h3> | + | <h3> Tolerance of <i><b>B. subtilis</b></i> against alkaline pH</h3> |
− | <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<sub>600</sub>) which correlates with bacterial growth. </p> | + | <p>The experiment to test the tolerance was performed as follows: <i>B. subtilis</i> 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<sub>600</sub>) which correlates with bacterial growth. </p> |
<figure class="makeresponsive" style="width: 70%; display: flex; align-items: center; justify-content: center; flex-direction: column;"> | <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/e/e3/T--TU_Dresden--bacillusinpH.png" | <img src="https://static.igem.org/mediawiki/2017/e/e3/T--TU_Dresden--bacillusinpH.png" | ||
Line 188: | Line 188: | ||
<p>The graph shows the growth of the organisms under each condition. It can be observed that from pH 7 to 9, no significant change in the growth is observed. However, under pH 10, the organism stops its growth. To check if the bacteria was still viable after the high pH treatment, it was transferred to growth plates with neutral pH. There, it was observed that <i>B. subtilis</i> was able to recover and growth normally, meaning that the organism can survive the process of encapsulation. </p> | <p>The graph shows the growth of the organisms under each condition. It can be observed that from pH 7 to 9, no significant change in the growth is observed. However, under pH 10, the organism stops its growth. To check if the bacteria was still viable after the high pH treatment, it was transferred to growth plates with neutral pH. There, it was observed that <i>B. subtilis</i> was able to recover and growth normally, meaning that the organism can survive the process of encapsulation. </p> | ||
<br> | <br> | ||
− | <h3> <i>Bacillus subtilis</i> in Peptidosomes</h3> | + | <h3> <i><b>Bacillus subtilis</b></i> in Peptidosomes</h3> |
<p>Once it was proven that <i>B. subtilis</i> can survive the designed encapsulation process, and that the organism cannot use the building block Fmoc-FF as a nitrogen source, experiments to entrap the bacteria started. </p> | <p>Once it was proven that <i>B. subtilis</i> can survive the designed encapsulation process, and that the organism cannot use the building block Fmoc-FF as a nitrogen source, experiments to entrap the bacteria started. </p> | ||
Line 225: | Line 225: | ||
<p>The encapsulation of <i>B. subtilis</i> expressing sfGFP in a constitutive way was also demonstrated by the fluorescence microscopy. In this experiment, the bacteria were encapsulated as described. As seen in Figure X, the peptidosome emitted green light what proved the existence of sfGFP-expressing bacteria. </p> | <p>The encapsulation of <i>B. subtilis</i> expressing sfGFP in a constitutive way was also demonstrated by the fluorescence microscopy. In this experiment, the bacteria were encapsulated as described. As seen in Figure X, the peptidosome emitted green light what proved the existence of sfGFP-expressing bacteria. </p> | ||
− | <h3> The Growth of <i>B.subtilis</i> in Peptidosomes</h3> | + | <h3> The Growth of <i><b>B.subtilis</b></i> in Peptidosomes</h3> |
<p>The method we used to check the growth and reproduction of bacteria inside the peptidosome was performed by generating peptidosomes loaded with a known amount of bacteria. Some peptidosomes were plated on LB agar right after being generated, while other peptidosomes were incubated in LB broth at 37°C for 3.5 and 7 hours and afterwards plated and incubated overnight. An increase in the number of colonies formed by the incubated peptidosomes means that bacteria can grow inside the structure. The result is shown in the next table. </p> | <p>The method we used to check the growth and reproduction of bacteria inside the peptidosome was performed by generating peptidosomes loaded with a known amount of bacteria. Some peptidosomes were plated on LB agar right after being generated, while other peptidosomes were incubated in LB broth at 37°C for 3.5 and 7 hours and afterwards plated and incubated overnight. An increase in the number of colonies formed by the incubated peptidosomes means that bacteria can grow inside the structure. The result is shown in the next table. </p> | ||
Line 263: | Line 263: | ||
<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> | ||
− | + | <br> | |
<figure class="makeresponsive" style="width: 65%; display: flex; align-items: center; justify-content: center; flex-direction: column;"> | <figure class="makeresponsive" style="width: 65%; display: flex; align-items: center; justify-content: center; flex-direction: column;"> | ||
<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." | ||
class="zoom"> | class="zoom"> | ||
− | <figcaption><b>Figure X: Dynabeads labeled with histidine-tagged GFP in the Binding/Wash buffer. </b> </figcaption> | + | <figcaption><b>Figure X: Dynabeads labeled with histidine-tagged GFP in the Binding/Wash buffer. </b> A) transmitted light B) fluorencence measurement </figcaption> |
</figure> | </figure> | ||
− | + | <br> | |
<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 (figure X). </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 (figure X). </p> | ||
+ | <br> | ||
<figure class="makeresponsive" style="width: 50%; display: flex; align-items: center; justify-content: center; flex-direction: column;"> | <figure class="makeresponsive" style="width: 50%; display: flex; align-items: center; justify-content: center; flex-direction: column;"> | ||
<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" |
Revision as of 12:41, 31 October 2017