diff --git a/static/style.css b/static/style.css
index 2429cfa3d59fa53fa878e7fec369e065de4f2655..0a15b2eca6ebfdd6e556eff8cc89781ba24f0e74 100644
--- a/static/style.css
+++ b/static/style.css
@@ -119,6 +119,32 @@ a[href^="#ref"] { /* Styling all hyperlinks */
width: 50%;
}
}
+
+/* Video styling */
+.video-container {
+ position: relative;
+ width: 560px; /* Default size for larger screens */
+ max-width: 100%; /* Ensures it adapts to smaller screens */
+ padding-top: 56.25%; /* 16:9 aspect ratio */
+ margin: 0 auto; /* Center align */
+ overflow: hidden;
+}
+
+.video-container iframe {
+ position: absolute;
+ top: 0;
+ left: 0;
+ width: 100%;
+ height: 100%;
+}
+
+/* Responsive adjustments */
+@media (max-width: 768px) {
+ .video-container {
+ width: 100%; /* Span the full width of smaller screens */
+ }
+}
+
/* -=-=-=-=-=-=-=-= Progress bar -=-=-=-=-=-=-=-= */
#progress-bar {
position: fixed;
diff --git a/wiki/pages/collaboration.html b/wiki/pages/collaboration.html
index 51f0611ecbb30caea5c4bf8d3ae7d37663f5f9b0..a564d4a86847450dbc703f49253052954f557aae 100644
--- a/wiki/pages/collaboration.html
+++ b/wiki/pages/collaboration.html
@@ -64,7 +64,7 @@
<div class="row mt-4" id="scavenger">
<h3 class="text-center">Protocol for Scavenger Hunt</h3>
- <img src="https://static.igem.wiki/teams/5250/team/collaborations/img-03-min.jpg" alt="Scavenger hunt" class="img-fluid" style="width: 50%; display: block; margin: 0 auto; padding: top 30px;">>
+ <img src="https://static.igem.wiki/teams/5250/team/collaborations/img-03-min.jpg" alt="Scavenger hunt" class="img-fluid" style="width: 50%; display: block; margin: 0 auto; padding: top 30px;">
</div>
<div class="row mt-4 justify-content-center">
diff --git a/wiki/pages/description.html b/wiki/pages/description.html
index 066f32dce8086d1b7b9792914ec64220ed4a1e11..0e995ba6ad159af1971798cc45bce45e1acf8275 100644
--- a/wiki/pages/description.html
+++ b/wiki/pages/description.html
@@ -143,9 +143,11 @@
<div class="row mt-4">
<div class="col-lg-12">
- <h2>Watch Our Presentation Video</h2>
- <hr>
- <iframe width="560" height="315" src="https://video.igem.org/videos/embed/b2a999ad-14d2-4a4e-b0cb-b9fc67681cf5" frameborder="0" allow="autoplay; encrypted-media" allowfullscreen></iframe>
+ <h2>Watch Our Presentation Video</h2>
+ <hr>
+ <div class="video-container">
+ <iframe src="https://video.igem.org/videos/embed/b2a999ad-14d2-4a4e-b0cb-b9fc67681cf5" frameborder="0" allow="autoplay; encrypted-media" allowfullscreen></iframe>
+ </div>
</div>
</div>
diff --git a/wiki/pages/experiments.html b/wiki/pages/experiments.html
index 6e82d779d7ea3eeb39bd361bb2aae725b916a9ff..c8be545eb0816045ddf411a103a23660194c363d 100644
--- a/wiki/pages/experiments.html
+++ b/wiki/pages/experiments.html
@@ -25,7 +25,7 @@
The positive control entails a plasmid that constitutively expresses GFP.
</p>
<p>
- Before starting the actual experiment we adjusted the cultures to OD 0.01 and into each well of a 96-well plate we added 20 μl of xylose and 180 μl of our adjusted culture .
+ Before starting the actual experiment we adjusted the cultures to OD 0.01 and into each well of a 96-well plate we added 20 μl of xylose and 180 μl of our adjusted culture.
In order to measure the fluorescence we used a plate reader.
We tested different xylose concentrations to find the optimum and to determine the amount of xylose the plant would need to exude in order to activate our promoter: 20mM, 10mM, 5mM, 2mM, 1mM, 0.5 mM, 0.2 mM, 0.1 mM, 0.01 mM.
We used a 24-hours program to measure OD and fluorescence every 30 minutes.
diff --git a/wiki/pages/model.html b/wiki/pages/model.html
index 2884821f29f28340a43b7312d1b0a0c77f2b15bc..3f3b1114241101f4cbb8650e01ef333620a6cf76 100644
--- a/wiki/pages/model.html
+++ b/wiki/pages/model.html
@@ -53,8 +53,9 @@ Holmes M, et al. (2003),", "title": "Pseudomonas putida KT2440 [ppu].", "link":
"https://www.sciencedirect.com/science/article/abs/pii/S0010482523002986?via%3Dihub"},
{"names": "[20] Purtschert-Montenegro, G., Cárcamo-Oyarce G., Pinto-Carbó M., Agnoli K., Bailly A., und Eberl L.",
"title": "„Pseudomonas Putida Mediates Bacterial Killing, Biofilm Invasion and Biocontrol with a Type IVB Secretion
-System“. Nature Microbiology 7, Nr. 10 (19. September 2022): 1547–57.", "link": "
-https://doi.org/10.1038/s41564-022-01209-6"}
+System“. Nature Microbiology 7, Nr. 10 (19. September 2022): 1547–57.", "link": "https://doi.org/10.1038/s41564-022-01209-6"},
+{"names": "[21] ",
+"title": "", "link": ""}
] %}
{% extends "layout_TOC.html" %}
@@ -1142,24 +1143,23 @@ https://doi.org/10.1038/s41564-022-01209-6"}
<h2>Update</h2>
<hr>
<p>
- In the time between wiki freeze and the Grand Jamboree, we continued tp work on our models and achived some
- more results, which we would like to present in this section.
+ Between the wiki freeze and the Grand Jamboree, we continued working on our models and achieved additional results, which we present in this section.
</p>
<p>
- We focused on the competition test mentioned in the <a href="#section-15" class="hyperlink-under">outlook</a> section.
- As a proof of concept, we chose two models of bacteria that our bacterium would most likely be competing against and decided to use one gram positive and one gram negative.
- For the gram positive we used models <i>Bacillus subtilis str. 168 </i>(iYO844) and for the gram negative <i>Xanthomonas phaseoli pv. manihotis </i>(iXpm1556).
- We adapted our pre-established soil conditions to these models and looked at the biomass production.
- Assuming that growth rate is a reasonable approximation of competitiveness, in the sense that a bacterium has an advantage if it can outgrow the others, we compared the levels at which the bacteria could grow biomass.
+ We focused on the competition test described in the <a href="#section-15" class="hyperlink-under">outlook</a> section.
+ As a proof of concept, we selected two GEMs of soil bacteria, a gram-positive and a gram-negative strain, that our bacterium would likely compete with in field applications.
+ For the gram-positive bacterium, we used <i>Bacillus subtilis</i> str. 168 and used its GEM, iYO844 <a href="#ref18">18</a>, and for the gram-negative bacterium, we chose <i>Xanthomonas phaseoli pv. manihotis </i>, using its GEM, iXpm1556<a href="#ref21">21</a>.
+ We adapted both models to our established soil conditions <a href="#section-11" class="hyperlink-under">in stage 3</a> and analyzed their biomass production in the given environment.
+ Assuming that growth rate is a reasonable proxy for competitiveness - where a bacterium gains a competitive advantage by outgrowing others - we compared their biomass production levels to those of our pre-established <i>P. putida</i> KT2440 model.
</p>
<div class="row mt-4 image-model">
<img src="https://static.igem.wiki/teams/5250/model/5-2-competitiontest-cell.webp"
alt="Dependence of Biomass Production on Cellulose Excretion in the Rhizosphere" class="img-fluid d-block mx-auto responsive-img">
- <p><b>Figure 24:</b> <i>P. putida</i> KT2440 shows a metabolic competition advantage to two other soil bacteria even when producing biofilm component cellulose.</p>
+ <p><b>Figure 24:</b> <i>P. putida</i> KT2440 shows a metabolic competition advantage to two other soil bacteria even when producing the biofilm component cellulose.</p>
</div>
<p>
- As shown in the graph, the <i>P. putida K2440</i> model outperforms both and can even be forced to produce cellulose before the cost becomes too high and the growth rate falls below that of the others.
- This result is very promising, but it needs to be followed up with more such tests and verification from the wet lab.
+ As shown in Figure 24, the <i>P. putida </i>KT2440 model outperforms both competitors and can even be induced to produce cellulose before the cost becomes too high and the growth rate falls below that of the others.
+ This result is very promising, but it requires further testing and verification/validation from the wet lab.
</p>
</div>
diff --git a/wiki/pages/new-basic-part.html b/wiki/pages/new-basic-part.html
index a5e98bee75d3fc245fb4c05ae15f215993bdd8aa..e47493e3e63edeea1858c0064a02dc5a25859920 100644
--- a/wiki/pages/new-basic-part.html
+++ b/wiki/pages/new-basic-part.html
@@ -19,7 +19,7 @@
<hr>
<div class="highlight">
<p>
- <b>We successfully modified a diguanylate cyclase (DGC) native to <i>Pseudomonas species</i> IsoF to stop c-di-GMP from binding to the negative allosteric binding site which effectively enhanced the enzyme’s activity.</b>
+ <b>We modified a diguanylate cyclase (DGC) native to <i>Pseudomonas species</i> IsoF to stop c-di-GMP from binding to the negative allosteric binding site which effectively enhanced the enzyme’s activity.</b>
</p>
</div>
<p>
@@ -63,7 +63,7 @@
<div class="row mt-0">
<div class="col">
<div class=image-model>
- <p style="text-align:center;"><b>Figure 2,3:</b>Fluorescence Intensity from c-di-GMP Assay across different Strains.</p>
+ <p style="text-align:center;"><b>Figure 2,3:</b> Fluorescence Intensity from c-di-GMP Assay across different Strains.</p>
</div>
</div>
</div>
diff --git a/wiki/pages/results.html b/wiki/pages/results.html
index 564fb4cd4b76b154f1e628a26d6161de4a39a541..ac354f9cdae84d2e815d03b278f246910f77921c 100644
--- a/wiki/pages/results.html
+++ b/wiki/pages/results.html
@@ -66,18 +66,17 @@
To optimize the protocol for the c-di-GMP assay, we conducted several preliminary measurements.
These pre-measurements aimed to identify the optimal OD and growth conditions to refine our experiment.
Our pre-measurements confirmed what we had previously heard from our experts: a low OD of 0.25 is optimal for this assay, whereas higher OD levels seem to lead to rapid oversaturation.
- Additionally, we tested two different media - LB medium and saline solution - and concluded that the saline solution produced more favorable experimental results.
+ Additionally, we tested two different media - LB medium and saline solution - and concluded that the saline solution produced more favorable results.
We observed that incubating the strains overnight with rhamnose resulted in poor gene expression, likely due to the depletion of rhamnose over time.
We determined that the optimal induction period for the cultures was up to 5 hours after adjusting them to an OD of 0.25.
</p>
- <!-- section id link!!!-->
<p>
- Built on these findings, we proceeded to conduct three c-di-GMP assays, focusing on the effects of PDE and DGC (<a href="#section-6" class="hyperlink-under">see section “Diguanylate cyclases“</a>) activity under the identified optimal conditions.
+ Built on these findings, we proceeded to conduct three c-di-GMP assays, focusing on the effects of PDE and Diguanylate cyclase (<a href="#section-6" class="hyperlink-under">see section “Diguanylate cyclases“</a>) activity under the identified optimal conditions.
</p>
<div class="image-model">
<img src="https://static.igem.wiki/teams/5250/project/results/img-01-1.png" alt=" Fluorescence Intensity from c-di-GMP Assay Across Different Strains, Experiment 1 and 2" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b> Figure 1: Fluorescence Intensity from c-di-GMP Assay across different Strains, Experiment 1 and 2:</b> The box plot shows the fluorescence intensity measurements for various <i>P. sp.</i> IsoF strains in a c-di-GMP assay, representing the c-di-GMP levels in each strain.
+ <b> Figure 1: Fluorescence Intensity from c-di-GMP Assay across different Strains, Experiment 1 and 2:</b> The box plot shows the fluorescence intensity measurements for various <span style="font-style: normal;">Pseudomonas sp.</span> IsoF strains in a c-di-GMP assay, representing the c-di-GMP levels in each strain.
</p>
</div>
<p>
@@ -137,8 +136,8 @@
In contrast, <i>P. sp.</i> IsoF YedQ displayed the highest fluorescence, reflecting the most polysaccharide production.
<i>P. sp.</i> IsoF dCas9 with the sgRNA showed higher fluorescence than the controls, but lower than <i>P. sp.</i> IsoF YedQ.
This suggests that the sgRNA inhibits the PDE PisoF_02645, leading to increased c-di-GMP levels and higher polysaccharide production.
- This is also shown in the raw data, as can be seen in figure 3. The table shows the mean fluorescence of the different strains.
- The strain containing the sgRNA and therefore inhibits the PDE on its genome, has increased fluorescence signal, and thus produces more polysaccharides, compared to the negative controls but shows less signal than the positive control.
+ This is also shown in the raw data, as can be seen in Table 1. The table shows the mean fluorescence of the different strains.
+ The strain contains the sgRNA and therefore inhibits the PDE on its genome, has increased fluorescence signal, and thus produces more polysaccharides, compared to the negative controls but shows less signal than the positive control.
The presence of the sgRNA alone leads to an increase in polysaccharide staining but as shown in the graph, inducing the strain containing the sgRNA with rhamnose did not result in a big difference compared to the non-induced strains.
Polysaccharide production did not increase substantially when dCas9 was induced compared to when it wasn’t, suggesting that the rhamnose promoter in front of dCas9 may be leaky.
Discussions with our advisor confirmed that similar results were observed in other gene knockdown experiments.
@@ -147,14 +146,14 @@
<div class="image-model">
<img src="https://static.igem.wiki/teams/5250/project/results/img-03-1.png" alt="Table with mean fluorescence" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 3: Table with mean fluorescence</b>
+ <b>Table 1: Table with mean fluorescence</b>
</p>
</div>
<p>
In the second biofilm staining assay we measured the strains containing the different DGCs and the strain containing the PDE knockdown.
All strains, including the controls, were once inoculated with rhamnose and once without rhamnose, to determine whether rhamnose itself affects biofilm component production.
Each strain and condition was tested in duplicates.
- The data, shown in Figure 4, shows similar results to the first assay in Figure 2.
+ The data, shown in Figure 3, shows similar results to the first assay in Table 1.
Fluorescence was higher in the <i>P. sp.</i> IsoF strain containing the sgRNA to knock down PisoF_02645 (labeled as “PDE knockdown†in this assay) than in the control strains.
While rhamnose induction caused a change in fluorescence, the increase was not as strong as in other induced strains.
Two different rhamnose promoters were used, one for the knockdown and one for the different DGCs.
@@ -169,7 +168,7 @@
<div class="row mt-4 image-description">
<img src="https://static.igem.wiki/teams/5250/project/results/img-04-1.png" alt="Biofilm staining assay 2 point plot" class="img-fluid " style="width: 50%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 4: Biofilm staining assay 2 point plot</b>
+ <b>Figure 3: Biofilm staining assay 2 point plot</b>
</p>
</div>
</div>
@@ -183,18 +182,18 @@
As PDEs degrade c-di-GMP, we expect to see less motility in strains with an overexpressed PDE and vice versa more in strains where the PDE is knocked down.
</p>
<p>
- Figure 5 shows that the knocked down PDE shows a decreased motility when rhamnose is added, as expected. With the PDE knocked down, c-di-GMP is not degraded, increasing the potential for biofilm formation, which aligns with our expectations.
+ Figure 4 shows that the knocked down PDE shows a decreased motility when rhamnose is added, as expected. With the PDE knocked down, c-di-GMP is not degraded, increasing the potential for biofilm formation, which aligns with our expectations.
</p>
<div class="row mt-4 image-description">
- <img src="https://static.igem.wiki/teams/5250/project/results/img-05-1.png" alt="Figure 5: Swimming Motility Assay point plot" class="img-fluid " style="width: 50%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/img-05-1.png" alt="Figure 4: Swimming Motility Assay point plot" class="img-fluid " style="width: 50%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 5: Swimming Motility Assay point plot</b>
+ <b>Figure 4: Swimming Motility Assay point plot</b>
</p>
</div>
<div class="highlight">
<p>
<b>Conclusion:</b>All three experiments confirm that PisoF_02645 was successfully knocked down, leading to an increased c-di-GMP concentration, enhanced polysaccharide production and restricted motility.
- Consequently, we achieved our goal of knocking down PisoF_02645 which in consequence enhances biofilm formation.
+ We achieved our goal of knocking down PisoF_02645 which in consequence enhances biofilm formation.
</p>
</div>
</div>
@@ -216,14 +215,14 @@
<h4>2.1 c-di-GMP assay</h4>
<div class="highlight">
<p>
- <b>Aim 1:</b> We want to prove that the introduction of a DGC can indeed increase biofilm production in <i>Pseudomonas sp.</i> IsoF.
+ <b>Aim 1:</b> We want to prove that the introduction of a DGC can indeed increase biofilm production in <i>P. sp.</i> IsoF.
</p>
<p>
<b>Aim 2:</b> We want to determine whether we can enhance the enzyme’s activity by modifying the allosteric binding site of c-di-GMP by mutating the sequence of PisoF_00565 by ourselves or by using the already established mutated sequence of WspR.
</p>
</div>
<p>
- To test our DGC constructs, we applied the same c-di-GMP assay as described for the PDE, with the findings illustrated in Figure 1.
+ To test our DGC constructs, we applied the same c-di-GMP assay as described for the PDE, with the findings illustrated in Figure 1 (shown again for clarity).
</p>
<div class="image-model">
<img src="https://static.igem.wiki/teams/5250/project/results/img-01-1.png" alt=" Fluorescence Intensity from c-di-GMP Assay Across Different Strains, Experiment 1 and 2" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
@@ -266,27 +265,27 @@
</p>
<p>
Next, we compared our constructs to a control <i>P. sp.</i> IsoF dCas9 without plasmid to quantify the change in c-di-GMP production.
- Several of our tested strains show a notable increase in c-di-GMP production compared to the control <i>P. sp.</i> IsoF dCas9 no plasmid, however only the mutated DGC WspR (DGC WspR R242A) showed statistical significance for it ( t = 2.4238, df = 7.3234, p-value = 0.04433).
- The high variability in the DGC WspR might suggest that while the construct has a promising potential to achieve high c-di-GMP levels, further optimization might be required to achieve more consistent results.
+ Several of our tested strains show a notable increase in c-di-GMP production compared to the control <i>P. sp.</i> IsoF dCas9 no plasmid, however only the mutated DGC WspR (DGC WspR R242A) showed statistical significance for it (t = 2.4238, df = 7.3234, p-value = 0.04433).
+ The high variability in the DGC WspR might suggest that while the construct has a promising potential to achieve high c-di-GMP levels, further optimization are required to achieve more consistent results.
Although our mutations of the DGC native to <i>P. sp.</i> IsoF (DGC PisoF R196A and DGC PisoF R240) did not show a significant increase in c-di-GMP levels compared to the empty plasmid, we believe that obtaining more data points could provide a clearer picture, as there appears to be a noticeable difference in c-di-GMP levels visually.
Further, when compared to <i>P. sp.</i> IsoF dCas9 empty plasmid, DGC PisoF R196A shows a significant increase (t = -2.9931, df = 5.3119, p-value = 0.02814) indicating inconsistencies in the data, since no difference between the two controls is expected.
</p>
<p>
A third experiment was excluded from the previous analysis because the values observed in the no-rhamnose condition were significantly different from those in the first two experiments.
This discrepancy suggested that the experiments might not be directly comparable, possibly due to unintended variations in the experimental setup.
- The results of the third experiment can be seen in Figure 6.
+ The results of the third experiment can be seen in Figure 5.
</p>
<div class="image-model">
- <img src="https://static.igem.wiki/teams/5250/project/results/img-06-1.png" alt="Figure 6: Fluorescence Intensity from c-di-GMP Assay Across Different Strains, Experiment 3." class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/img-06-1.png" alt="Figure 5: Fluorescence Intensity from c-di-GMP Assay Across Different Strains, Experiment 3." class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b> Figure 6: Fluorescence Intensity from c-di-GMP Assay Across Different Strains, Experiment 3.</b>
+ <b> Figure 5: Fluorescence Intensity from c-di-GMP Assay Across Different Strains, Experiment 3.</b>
</p>
</div>
<p>
Since there was only one data point per strain and condition, no statistical test was conducted.
Differences to the empty plasmid are only visible for the two mutated DGCs, DGC PisoF R196A and DGC PisoF R240A.
The difference in fluorescence signal of PisoF R196A compared to the empty plasmid is 239.00 and 329.00 for DGC PisoF R24 compared to the empty plasmid respectively.
- This supports the findings from the data shown in figure 1.
+ This supports the findings from the data shown in Figure 1.
Introducing a DGC elevates c-di-GMP concentrations and again, it seems like the mutation of PisoF_00565 at position 196 worked and the modified enzyme seems to have increased activity.
</p>
<p>
@@ -301,15 +300,15 @@
<div class="row mt-4">
<h4>2.2 Biofilm staining</h4>
<p>
- Figure 4 represents the fluorescence data of the strains containing the five different DGCs (alongside the strain containing the sgRNA to knockdown PDE).
+ Figure 3 represents the fluorescence data of the strains containing the five different DGCs (alongside the strain containing the sgRNA to knockdown PDE).
The figure illustrates the mean fluorescence for each strain.
<i>P. sp.</i> IsoF dCas9 and <i>P. sp.</i> IsoF with an empty pBBR1MCS5 plasmid served as negative controls, while <i>P. sp.</i> IsoF YedQ once again served as a positive control.
Each strain and condition (with and without rhamnose) was measured in duplicate.
</p>
<div class="image-description">
- <img src="https://static.igem.wiki/teams/5250/project/results/img-04-1.png" alt="Biofilm staining assay 2 point plot" class="img-fluid " style="width: 50%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/img-04-1.png" alt="Figure 3: Biofilm staining assay 2 point plot" class="img-fluid " style="width: 50%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 4: Biofilm staining assay 2 point plot</b>
+ <b>Figure 3: Biofilm staining assay 2 point plot</b>
</p>
</div>
<p>
@@ -361,7 +360,7 @@
Surprisingly, the mutated forms of DGC PisoF_00565, R196A and R240A did not lead to a notable increase in polysaccharide production.
</p>
<p>
- These results confirm that introducing certain diguanylate cyclases (DGCs) can significantly increase and therefore promote polysaccharide production, which represents an enhanced and stronger biofilm formation.
+ These results confirm that introducing certain DGCs can significantly increase and therefore promote polysaccharide production, which represents an enhanced and stronger biofilm formation.
Elevating polysaccharide production through the introduction of a DGC provides an efficient and straightforward approach to boost biofilm formation, without the need to focus only on specific genes in the genetic pathway to produce one or two polysaccharides.
</p>
</div>
@@ -372,33 +371,33 @@
As DGCs drive biofilm formation by producing c-di-GMP, we expect to see a shift in our DGC strains from a motile to a more sessile state in which biofilm-formation can take place.
</p>
<p>
- Figure 5 shows that the DGC WspR and the mutated form WspR R242A show the highest motility (indicated by the swimming motility diameter) when the DGC is not induced by rhamnose.
+ Figure 4 shows that the DGC WspR and the mutated form WspR R242A show the highest motility (indicated by the swimming motility diameter) when the DGC is not induced by rhamnose.
The high motility levels suggest that these strains may be in a state with less biofilm formation.
However, in the presence of rhamnose, their motility drastically decreases in the first assay (the effect is not as drastic in the second one).
This indicates that when c-di-GMP is further increased due to DGC activity, motility decreases as expected, which aligns with an increased potential for biofilm formation.
The sharp drop in motility suggests that WspR likely contributes heavily to biofilm formation when expressed.
</p>
<div class="image-description">
- <img src="https://static.igem.wiki/teams/5250/project/results/img-05-1.png" alt="Figure 5: Swimming Motility Assay point plot" class="img-fluid " style="width: 50%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/img-05-1.png" alt="Figure 4: Swimming Motility Assay point plot" class="img-fluid " style="width: 50%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 5: Swimming Motility Assay point plot</b>
+ <b>Figure 4: Swimming Motility Assay point plot</b>
</p>
</div>
<p>
Our mutations DGC PIsoF R240A and R196A show lower motility in both conditions, yet the rhamnose condition shows higher motility than no rhamnose condition.
This suggests that this DGC may not be as important as the WspR in decreasing the motility for example.
- Our controls <i>P. sp. IsoF</i> no plasmid, empty plasmid and PDE overexpression show higher levels than our rhamnose induced DGCs.
+ Our controls <i>P. sp. </i>IsoF no plasmid, empty plasmid and PDE overexpression show higher levels than our rhamnose induced DGCs.
</p>
<p>
These results confirm that for some DGCs, such as DGC WspR and DGC WspR R242A, the relationship between DGC activity, motility, and the biofilm formation process is strongly correlated.
- While it is not clear why our DGC mutations R196A and R240A show higher motility with rhamnose, this is also seen when rhamnose is added to the YedQ DGC.
+ While it is not clear why our DGC mutations R196A and R240A show higher motility with rhamnose, this is also seen when rhamnose is added to the DGC YedQ.
This may suggest that rhamnose could promote bacterial motility in other metabolic contexts, separate from DGCs.
</p>
<div class="highlight">
<p>
<b>Conclusion:</b>
- We successfully achieved our goal of promoting biofilm formation through the implementation of DGCs in <i>P. sp. IsoF</i> .
- Introducing a DGC into <i>P. sp. IsoF</i> increased intracellular c-di-GMP levels, enhanced the production of polysaccharides and reduced bacterial motility.
+ We successfully achieved our goal of promoting biofilm formation through the implementation of DGCs in <i>P. sp. </i>IsoF.
+ Introducing a DGC into <i>P. sp. </i>IsoF increased intracellular c-di-GMP levels, enhanced the production of polysaccharides and reduced bacterial motility.
All key factors for a robust biofilm formation.
These findings suggest that the introduction of a DGC effectively enhances biofilm formation.
Elevating intracellular c-di-GMP levels through the introduction of a DGC therefore provides an efficient and straightforward approach to boost biofilm formation by regulating different genes, without the need to focus only on specific genes in the pathway.
@@ -411,7 +410,7 @@
<hr>
<h3></h3>
<div class="highlight">
- <p><b>Aim:</b> We inoculated plant roots with three different bacterial strains of Pseudomonas species IsoF and with LB medium to assess their effects on the root and shoot weight in normal and drought conditions.</p>
+ <p><b>Aim:</b> We inoculated plant roots with three different bacterial strains of <i>P. sp.</i> IsoF and with LB medium to assess their effects on the root and shoot weight in normal and drought conditions.</p>
</div>
<p>
Raw data from the plant experiment was obtained on the date of inoculation - time point 0, and two weeks later.
@@ -423,35 +422,35 @@
</p>
<h4>Time point 0</h4>
<p>
- One plant out of each treatment for the dry and wet conditions was taken out and processed on the first day after 30 minutes of inoculation.
+ One plant out of each treatment for the drought and normal/watered conditions was taken out and processed on the first day after 30 minutes of inoculation.
The initial measurements are summarized in the following graph:
</p>
<div class="image-model">
- <img src="https://static.igem.wiki/teams/5250/project/results/img-07.png" alt="Figure 7: Initial measurements of the plants at time point 0 (30 minutes after inoculation)" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/img-07.png" alt="Figure 6: Initial measurements of the plants at time point 0 (30 minutes after inoculation)" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 7: Initial measurements of the plants at time point 0 (30 minutes after inoculation)</b>
+ <b>Figure 6: Initial measurements of the plants at time point 0 (30 minutes after inoculation)</b>
The net weights of the wet and dry aerial part, as well as the net weight of the rhizosphere is included in the measurements.
</p>
</div>
<p>
- Figure 7 represents the time point 0 measurements that serve as a baseline for our analysis. Based on these values we can make observations about the growth that the rest of the plants in the same treatment group exhibit.
+ Figure 6 represents the time point 0 measurements that serve as a baseline for our analysis. Based on these values we can make observations about the growth that the rest of the plants in the same treatment group exhibit.
</p>
<h4>Wet weight Measurement after 14 days</h4>
<p>We conducted the second measurement after 14 days. The remaining plants from each treatment in the dry or wet conditions respectively were processed.
- The data points are depicted in Figure 8:
+ The data points are depicted in Figure 7:
</p>
<div class="image-model">
- <img src="https://static.igem.wiki/teams/5250/project/results/img-08.png" alt="Figure 8: Net dry and wet weight of the treatment groups under normal and drought conditions." class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/img-08.png" alt="Figure 7: Net dry and wet weight of the treatment groups under normal and drought conditions." class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 8: Net dry and wet weight of the treatment groups under normal and drought conditions.</b>
- In green is the net wet weight of all plants processed at time point 0.
+ <b>Figure 7: Net dry and wet weight of the treatment groups under normal and drought conditions.</b>
+ In green is the net wet weight of all plants processed at Time point 0.
</p>
</div>
<p>
As expected, the drought conditions result in an overall lower mass of the aerial part of the plant than the normal conditions.
However, under drought conditions there is barely a difference in the mean values between the treatments.
This contradicts our hypothesis that the bacterial treatments would result in a higher biomass than the LB medium treatment.
- We think this might be due to the dry treatment being too harsh, possibly resulting in conditions too dry for the bacteria to have any beneficial effect.
+ We think this might be due to the drought treatment being too harsh, possibly resulting in conditions too dry for the bacteria to have any beneficial effect.
In addition, the variance in the data points we obtained from the LB treatment was too high for us to effectively draw a conclusion from it.
The same applies to all the data points obtained from the wet measurements.
</p>
@@ -470,13 +469,13 @@
<p>
After we processed the wet weight, we kept the plants in the oven for a few days until they were completely dry.
Then we measured them again to obtain the dry weight of all plants.
- These measurements are illustrated in Figure 9 below:
+ These measurements are illustrated in Figure 8 below:
</p>
<div class="image-model">
- <img src="https://static.igem.wiki/teams/5250/project/results/img-09.png" alt="Figure 9: Net dry weight of the aerial part of the treatment groups under normal and drought conditions." class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/img-09.png" alt="Figure 8: Net dry weight of the aerial part of the treatment groups under normal and drought conditions." class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 9: Net dry weight of the aerial part of the treatment groups under normal and drought conditions.</b>
- In green is the net dry weight of all plants processed at time point 0.
+ <b>Figure 8: Net dry weight of the aerial part of the treatment groups under normal and drought conditions.</b>
+ In green is the net dry weight of all plants processed at Time point 0.
</p>
</div>
<p>
@@ -495,32 +494,36 @@
However, there is high variability between the individual data points,therefore no meaningful observation can be made about the effect of the different treatment groups.
For future experiments and better statistical analysis, we would need to include more plants in our treatment groups.
</p>
+ <p>
+ Since the dry weight, but not the wet weight, increases with YedQ, we can infer that YedQ—and consequently, enhanced biofilm formation—improves water retention potential.
+ With a more optimized experimental setup, such as an extended growth period and younger, more actively growing plants, this effect might more effectively translate into improved plant growth.
+ </p>
<h4>CFU count</h4>
<p>
We conducted a colony forming unit count from the rhizosphere of each of the treated plants.
With the aim of establishing the number of <i>P. sp.</i> IsoF colonies 14 days after the treatment implementation we took a sample of the rhizosphere and performed a dilution series.
- Those were plated and counted.
- Different amounts of soil were stuck to the roots in each sample, we adjusted the count for 1g of rhizosphere.
+ The diluted samples were plated and counted.
+ Different amounts of soil were stuck to the roots in each sample and we adjusted the count for 1g of rhizosphere.
</p>
<div class="image-model">
- <img src="https://static.igem.wiki/teams/5250/project/results/img-10.png" alt="Figure 10: CFU/g rhizosphere from plants extracted at Timpoint 0" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/img-10.png" alt="Figure 9: CFU/g rhizosphere from plants extracted at Timpoint 0" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 10: CFU/g rhizosphere from plants extracted at Timpoint 0</b>
+ <b>Figure 9: CFU/g rhizosphere from plants extracted at Timpoint 0</b>
</p>
</div>
<p>
- Figure 10 shows that the DGC YedQ and PDE overexpression strains have the highest number of surviving colonies, followed by the PisoF pBBR1MCS5 plasmid and LB.
+ Figure 9 shows that the DGC YedQ and PDE overexpression strains have the highest number of surviving colonies, followed by the PisoF pBBR1MCS5 plasmid and LB.
The plates we used had a gentamicin resistance to help us select for our strains that carry the plasmids we introduced with a gentamicin resistance.
This aligns with our expectations and proves that <i>P. sp.</i> IsoF can survive in conditions mimicking the environment and retain the plasmids that have been introduced to it.
The difference between the wet and dry conditions can be explained by the fact that some of the bacteria might have been washed away when the wet plants were watered.
</p>
<p>
- In addition, the data we obtained 14 days after inoculation represented in figure 11 shows more relevant results.
+ In addition, the data we obtained 14 days after inoculation represented in Figure 10 shows more relevant results.
</p>
<div class="image-model">
- <img src="https://static.igem.wiki/teams/5250/project/results/img-11.png" alt="Figure 11: CFU/g rhizosphere from plants extracted after 14 days of growth" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/img-11.png" alt="Figure 10: CFU/g rhizosphere from plants extracted after 14 days of growth" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 11: CFU/g rhizosphere from plants extracted after 14 days of growth</b>
+ <b>Figure 10: CFU/g rhizosphere from plants extracted after 14 days of growth</b>
</p>
</div>
<p>
@@ -538,7 +541,7 @@
<b>Conclusion:</b>
It can be said that while we did not observe a substantial increase in the biomass of the treated plants that underwent drought conditions, the results we observed from the regularly watered plants supported our main hypothesis - the enhanced biofilm of <i>P. sp.</i> IsoF is beneficial for plant growth.
Moreover, our CFU count experiment confirmed that our biofilm overexpression modules do not cause more of a metabolic strain on the bacteria in conditions mimicking the environment than the empty plasmid.
- However, more samples are needed overall for future plant experiments for us to be able to conduct proper statistical tests and come to final conclusions.
+ However, more samples are needed overall to conduct proper statistical tests and come to final conclusions.
</p>
</div>
</div>
@@ -560,17 +563,16 @@
<h3>Toxin Assay 1: OD Measurement</h3>
<h4>Toxin assay 1 in <i>E. coli</i> SY327 (1)</h4>
<div class="image-model">
- <img src="https://static.igem.wiki/teams/5250/project/results/ks1.png" alt="Figure 12" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/ks1.png" alt="Figure 11" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 12:</b>
- As negative control we used <i>E. coli</i> SY327 with an empty backbone of the same plasmids used for our toxins (pJUMP29-1D(sfGFP)).
+ <b>Figure 11:</b>
+ As a negative control we used <i>E. coli</i> SY327 with an empty backbone of the same plasmids used for our toxins (pJUMP29-1D(sfGFP)).
The three strengths (weak, medium and strong) refer to the strengths of the RBSs associated with the toxins in the respective strain (pT41Rhyz01, pT41Rhyz02, pT41Rhyz03).
We inoculated the strains with either 1% rhamnose or no rhamnose (+0%).
</p>
</div>
<p>
The toxin test did not work, as there was no significant difference in cell growth between the cells induced with rhamnose and the ones that grew without rhamnose.
- If anything, the cells treated with rhamnose seemed to grow better, potentially using rhamnose as an extra source of energy.
</p>
<p>
Before testing our toxin again, we needed to rule out that the issue lies with the rhamnose promoter.
@@ -587,16 +589,16 @@
To quantify the promoter’s activity at different rhamnose concentrations and the potentially toxic or nutritious effect of rhamnose on the cell, we performed an overnight growth assay, measuring OD600 and fluorescence at excitation 530±25 nm and emission 590±35 nm.
</p>
<div class="image-model">
- <img src="https://static.igem.wiki/teams/5250/project/results/ks2.png" alt="Figure 13" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/ks2.png" alt="Figure 12" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 13:</b>
+ <b>Figure 12:</b>
Depicted is the fluorescence, and thus promoter activity at different rhamnose concentrations (1%, 1.5%, 2%, 2.5% and 3%). The values are adjusted to an OD600 of 1.00 to account for the fluorescence being stronger at higher cell numbers.
We additionally measured the autofluorescence of <i>E. coli</i> SY327 wildtype when inoculated with the same rhamnose concentrations.
We subtracted said autofluorescence from the promoter activity data to isolate the fluorescence obtained from the RFP production alone.
</p>
</div>
<p>
- The promoter was detectably activated after 2hrs of inoculation with rhamnose, regardless of concentration.
+ The promoter was detectably activated after 2hrs of inoculation with rhamnose, regardless of the concentration.
The highest fluorescence per OD and thus the strongest promoter activity was at 1% rhamnose, followed by 1.5%.
This means the promoter’s maximum activity with the minimum negative effect of rhamnose was at 1%.
</p>
@@ -605,9 +607,9 @@
</p>
<h4>Toxin Testing in <i>E. coli</i> SY327 (2)</h4>
<div class="image-model">
- <img src="https://static.igem.wiki/teams/5250/project/results/ks3.png" alt="Figure 14" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/ks3.png" alt="Figure 13" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 14:</b>
+ <b>Figure 13:</b>
Displayed are the OD600 of <i>E. coli</i> SY327 wildtype (wt) and said strain with an empty backbone (empty plas) of the same plasmids used for our toxins (pJUMP29-1D(sfGFP)) at 0%, 1% and 1.5% rhamnose.
As a positive control (pos cont), we added Kanamycin to the wildtype strain which is sensitive to this antibiotic.
The three strengths (weak, medium and strong) refer to the strengths of the RBSs associated with the toxins in the respective strain (pT41Rhyz01, pT41Rhyz02, pT41Rhyz03).
@@ -615,15 +617,13 @@
</div>
<p>
In this second toxin test on <i>E. coli</i> SY327, we again observed no significant difference in the strains’ growth between the ones with an induced toxin production and the ones without.
- Generally, the growth did not differ between the toxin strengths and the controls, but rather reflected the changes in rhamnose concentrations.
- Generally, most strains that grew the most were the ones with no rhamnose added, followed by 1% and then 1.5%. We concluded that rhamnose is affecting the cells’ growth negatively and more so than the toxin, regardless of its strength.
- To account for that, we subtracted the growth of the empty plasmid control (pJUMP29-1D(sfGFP)) from the strains tested at the respective rhamnose concentrations.
+ We subtracted the growth of the empty plasmid control (pJUMP29-1D(sfGFP)) from the strains tested at the respective rhamnose concentrations.
This way, we would see the difference in growth caused by our toxin plasmid alone - expecting at least some slowed growth.
</p>
<div class="image-model">
- <img src="https://static.igem.wiki/teams/5250/project/results/ks4.png" alt="Figure 15" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/ks4.png" alt="Figure 14" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 15:</b>
+ <b>Figure 14:</b>
We subtracted the growth of the empty plasmid control (pJUMP29-1D(sfGFP)) from the strains tested at the respective rhamnose concentrations (0%, 1% and 1.5%).
This way, we see the difference in growth caused by our toxin plasmid alone.
The three strengths (weak, medium and strong) refer to the strengths of the RBSs associated with the toxins in the respective strain (pT41Rhyz01, pT41Rhyz02, pT41Rhyz03).
@@ -636,16 +636,16 @@
</p>
<p>
We concluded from this that the most likely cause for our toxin’s lack of effect is a gyrase mutation in <i>E. coli</i> SY327.
- Upon further research and consultation with Zaira we found that <i>E. coli</i> SY327 is based on <i>E. coli</i> S17-1, which carries a gyrA462 allele - a gyrase mutation making cells immune to ccdB toxins<a href="#ref3">3</a>.
+ Upon further research and consultation with Dr. Zaira Heredia Ponce we found that <i>E. coli</i> SY327 is based on <i>E. coli</i> S17-1, which carries a gyrA462 allele - a gyrase mutation making cells immune to ccdB toxins<a href="#ref3">3</a>.
This allele has been deliberately transferred to several strains derived from <i>E. coli</i> S17-1, though we could not find evidence to support it was also transferred to <i>E. coli</i> SY327.
Still, we found this to be reason enough to assume our current <i>E. coli</i> strain had a gyrA mutation, wielding it immune to the ccdB toxin and thus explaining our toxin’s lack of effect. We therefore had to transform our plasmids into a new strain and repeat the toxin test.
After some brief research and consulting which strains were readily available by our host lab, we chose to go with <i>E. coli</i> DH5α.
</p>
<h4>Toxin assay 1 in <i>E. coli</i> DH5α</h4>
<div class="image-model">
- <img src="https://static.igem.wiki/teams/5250/project/results/ks5.png" alt="Figure 16" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/ks5.png" alt="Figure 15" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 16:</b>
+ <b>Figure 15:</b>
Shown is the OD600 of <i>E. coli</i> DH5α with the toxin strengths and a control at respective rhamnose concentrations (0%, 1% and 1.5%).
As a control (GFP plas) we used a plasmid consisting of the same backbone as our toxins but with GFP, to have a comparable metabolic strain of the toxin, but without its toxic effect (p03Rhyz11).
As a positive control (pos cont), we added Kanamycin to the GFP plas strain which is sensitive to this antibiotic.
@@ -656,15 +656,14 @@
Once more, the cell’s growth seems to be unaffected by the ccdB toxin, as no significant difference was found between the control, the different toxin strengths, or their induction with rhamnose.
We revisited our plasmid construct to eliminate all possibilities of a design error.
While in our original research we found no gyrA mutations in <i>E. coli</i> DH5α, an AddGene blog entry by Mathew Ferenc shed light on a gyrase mutation (gyrA96) mutation carried by <i>E. coli</i> DH5α which, though not proven to provide resistance to ccdB, may affect the toxin’s effectivity.<a href="#ref4">4</a>
- While this finding was frustrating, it taught us how unorganized and hidden scientific information can be - the lack of one universal database for bacterial strains just being one example.
- We used Ferenc’s blog to screen for other potentially sensitive <i>E. coli</i>strains and noted the strains which our lab had available.
+ We used Ferenc’s blog to screen for other potentially sensitive <i>E. coli</i> strains and noted the strains which our lab had available.
We decided to continue the testing of our toxin in HB101, MC1061 and CSH50.
</p>
<h4>Toxin assay 1 in <i>E. coli</i> HB101, <i>E. coli</i> MC1061 and <i>E. coli</i> CSH50</h4>
<div class="image-description">
- <img src="https://static.igem.wiki/teams/5250/project/results/ks6.png" alt="Figure 17" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/ks6.png" alt="Figure 16" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 17:</b>
+ <b>Figure 16:</b>
Shown is the OD600 of <i>E. coli</i> HB101 with the toxin strengths and a control at respective rhamnose concentrations (0%, 1% and 1.5%). As a control (GFP plas) we used a plasmid consisting of the same backbone as our toxins but with GFP, to have a comparable metabolic strain of the toxin, but without its toxic effect (p03Rhyz11).
We also tested the <i>E. coli</i> HB101 wildtype strain (wt) to observe the effects of rhamnose on it.
As a positive control (pos cont), we added Kanamycin to the wildtype strain which is sensitive to this antibiotic.
@@ -672,9 +671,9 @@
</p>
</div>
<div class="image-description">
- <img src="https://static.igem.wiki/teams/5250/project/results/ks7.png" alt="Figure 18" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/ks7.png" alt="Figure 17" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 18:</b>
+ <b>Figure 17:</b>
We measured the OD600 of <i>E. coli</i> MC1061 with the toxin strengths and a control at respective rhamnose concentrations (0%, 1% and 1.5%).
As a control (wt) we used the <i>E. coli</i>MC1061 wildtype strain.
As a positive control (pos cont), we added Kanamycin to the wildtype strain which is sensitive to this antibiotic.
@@ -682,9 +681,9 @@
</p>
</div>
<div class="image-description">
- <img src="https://static.igem.wiki/teams/5250/project/results/ks8.png" alt="Figure 19" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/ks8.png" alt="Figure 18" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 19:</b>
+ <b>Figure 18:</b>
Shown is the OD600 of <i>E. coli</i> CSH50 with the toxin strengths and a control at respective rhamnose concentrations (0%, 1% and 1.5%).
As a control (wt) we used the <i>E. coli</i> CSH50 wildtype strain.
As a positive control (pos cont), we added Kanamycin to the wildtype strain which is sensitive to this antibiotic.
@@ -695,15 +694,15 @@
In all three <i>E. coli</i> strains tested above (HB101, MC1061 and CSH50), the cell’s growth seems to be unaffected by the ccdB toxin, as no significant difference was found between the controls, the different toxin strengths, or their induction with rhamnose.
There were still some differences between the tests such as <i>E. coli</i> MC1061 strains having more variability, <i>E. coli</i> HB101 overall growing to higher concentrations and the positive control in <i>E. coli</i> CSH50 seemingly growing slightly despite the presence of Kanamycin.
However, none of these differences in results affect the conclusion that our ccdB toxin system does not kill or inhibit any of our cell’s growth.
- As at this stage of our project we were starting to run out of time, we decided to perform a last toxin test in <i>Pseudomonas species</i> IsoF for the sake of thoroughness.
+ As at this stage of our project we were starting to run out of time, we decided to perform a last toxin test in <i>P. sp.</i> IsoF for the sake of thoroughness.
</p>
<h4>Toxin Testing in <i>P. sp.</i> IsoF</h4>
<div class="image-description">
- <img src="https://static.igem.wiki/teams/5250/project/results/ks9.png" alt="Figure 120" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/ks9.png" alt="Figure 19" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
<p style="text-align:center;">
- <b>Figure 20:</b>
- Shown is the OD600 of <i>P. sp.</i> IsoF with the different toxin strengths and a control at respective rhamnose concentrations (0%, 1% and 1.5%).
- As a control (empty plas) we used the <i>P. sp.</i> IsoF with the same backbone from the toxin-carrying strains (pBBR1MCS5).
+ <b>Figure 19:</b>
+ Shown is the OD600 of <span style="font-style: normal;">P. sp.</span> IsoF with the different toxin strengths and a control at respective rhamnose concentrations (0%, 1% and 1.5%).
+ As a control (empty plas) we used the <span style="font-style: normal;">P. sp.</span> IsoF with the same backbone from the toxin-carrying strains (pBBR1MCS5).
As a positive control (pos cont), we added Kanamycin to the wildtype strain which is sensitive to this antibiotic.
The three strengths (weak, medium and strong) refer to the strengths of the RBSs associated with the toxins in the respective strain (pBBR pT41Rhyz01, pBBR pT41Rhyz02, pBBR pT41Rhyz03).
</p>
@@ -718,7 +717,6 @@
We’ve decided to incorporate this method into our next experiment.
</p>
<h3>TOXIN ASSAY 2: rhamnose induced plates</h3>
- <h4>Toxin assay 2 in <i>P. sp.</i> IsoF</h4>
<p>
As mentioned earlier, we adjusted our toxin testing protocol by incorporating rhamnose into the plates, with the goal of performing a CFU (colony-forming unit) assay.
</p>
@@ -727,24 +725,48 @@
<div class="row mt-0">
<div class="col-lg-6 col-md-6 col-sm-12 order-1 order-md-1">
<img src="https://static.igem.wiki/teams/5250/project/results/ks-10-min.jpg"
- alt="Figure 21" class="img-fluid" style="width: 100%;">
+ alt="Figure 20" class="img-fluid" style="width: 100%;">
</div>
<div class="col-lg-6 col-md-6 col-sm-12 order-2 order-md-2">
- <img src="https://static.igem.wiki/teams/5250/project/results/ks-11-min.jpg" alt="Figure 21" class="img-fluid" style="width: 100%;">
+ <img src="https://static.igem.wiki/teams/5250/project/results/ks-11-min.jpg" alt="Figure 20" class="img-fluid" style="width: 100%;">
</div>
</div>
<div class="row mt-0">
<div class="image-description">
- <p style="text-align:center;"><b>Figure 21:</b> Comparison of <i>E. coli</i> CSH50 PT41Rhyz03 (left) and <i>P. sp.</i> IsoF (right) grown on agar plates containing varying rhamnose concentrations (0%, 1%, and 1.5%).</p>
+ <p style="text-align:center;"><b>Figure 20:</b> Comparison of <span style="font-style: normal;">E. coli</span> CSH50 PT41Rhyz03 (left) and <span style="font-style: normal;">P. sp.</span> IsoF (right) grown on agar plates containing varying rhamnose concentrations (0%, 1%, and 1.5%).</p>
</div>
</div>
<div class="row mt-4">
<p>
- Figure 21 shows the plates after letting the bacteria grow over the weekend. Unfortunately, we did not prepare a dilution series, making it difficult to accurately analyze the results.
- Due to time constraints, we were unable to repeat the experiment with a proper dilution series. However, based on visual inspection of the plates, we were not satisfied with the results and could not conclude that our toxin had a clear impact on reducing CFU.
+ Figure 20 shows the plates after letting the bacteria grow over the weekend.
+ Based on visual inspection of the plates we determined that there is an inhibition of growth in <i>E. coli</i> CSH50 strain but not in the <i>P. sp.</i> IsoF strain.
+ However, the CFU count in some of the plates was too high for us to effectively count.
+ Therefore, we decided to conduct 2 more repeats of the toxin assay with a starting OD600 reduced tenfold to obtain quantitative data.
+ The results are summarized in the following histogram:
+ </p>
+ <div class="image-description">
+ <img src="https://static.igem.wiki/teams/5250/project/results/copy-of-graph-toxin-assay-2.webp" alt="Figure 21" class="img-fluid " style="width: 75%; display: block; margin-left: auto; margin-right: auto;">
+ <p style="text-align:center;">
+ <b>Figure 21:</b>
+ CFU count of the <span style="font-style: normal;">E. coli.</span> CSH50 strain and <span style="font-style: normal;">P. sp.</span> IsoF strain containing the toxin plasmid against a control strain of both bacterial species containing an empty plasmid.
+ </p>
+ </div>
+ <p>
+ Figure 21 demonstrates a lower CFU count in the <i>E. coli</i> CSH50 plates expressing the CcdB toxin.
+ Compared to the control strain containing the empty plasmid, we can conclude that the toxin effectively inhibits growth in <i>E. coli</i>.
+ There is a tenfold decrease in the CFU count in the <i>E. coli</i> 1.5% rhamnose plate compared to the 0% rhamnose plate.
+ Another observation we made is that the growth inhibition is dependent on the concentration of rhamnose we used in the plates - the higher the rhamnose concentration, the lower the CFU count.
+ However, there is no visible effect from the toxin on the <i>P. sp.</i> IsoF strain.
+ Both the control strain and the toxin expressing strain give identical results.
</p>
+ <p>
+ We undertook various troubleshooting measures, including testing different strains that lacked resistance, evaluating the functionality of our rhamnose promoter, examining our construct, and experimenting with different conditions.
+ Eventually we were able to show that the toxin we used inhibits growth in <i>E. coli</i>.
+ Nevertheless, due to time constraints we were not able to test whether the CcdA antitoxin neutralizes the toxin activity.
+ </p>
+
<div class="highlight">
<p><b>Conclusion:</b> Despite our extensive efforts, we were unable to establish a functional kill switch.</p>
</div>
@@ -780,13 +802,11 @@
</p>
<h5>Kill Switch:</h5>
<p>
- We will conduct another toxin experiment in which we directly expose colonies to the ccdB protein.
- If the bacteria are killed under these conditions, it would support our suspicion that the promoter may not be strong enough to produce enough toxin.
Instead of redoing another toxin experiment with the CcdB/CcdA system, that has initially been established for <i>E. coli</i> strains, we could switch to another toxin-antitoxin system, that is established in <i>Pseudomonas species</i>.
</p>
<h5>Final construct:</h5>
<p>
- If we had more time we would have recloned the level 2 plasmids and introduced them into P. sp. IsoF to test our genetic circuit.
+ If we had more time we would have recloned the level 2 plasmids and introduced them into <i>P. sp.</i> IsoF to test our genetic circuit.
</p>
<p>
We would first research the concentration of xylose exceeded by plant roots.
diff --git a/wiki/pages/safety.html b/wiki/pages/safety.html
index 10a41f51010c67ba562fe55da1c776915deaebb7..ca077b6640210af9929fc7bcd7e3ff879ea6862a 100644
--- a/wiki/pages/safety.html
+++ b/wiki/pages/safety.html
@@ -67,7 +67,7 @@
<p>
At the current stage, the nationwide moratorium on GMOs in Switzerland would significantly complicate the field testing and application of our product<a href="#ref3">3</a>.
However, as of 2024, the longevity of this law is under discussion.
- To find out more about the implications of the moratorium on our project, visit our <a href="{{ url_for('pages', page='entrepreneurship') }}" class="hyperlink-under">Implementation and Entrepreneurship page</a>.
+ To find out more about the implications of the moratorium on our project, visit our <a href="{{ url_for('pages', page='entrepreneurship') }}" class="hyperlink-under">Entrepreneurship and Implementation page</a>.
Regardless, our product accounts for a safe application and prevents a release beyond containment through several approaches:
</p>
</div>
@@ -81,10 +81,10 @@
<div class="row">
<div class="col-12">
<p><b>Killswitch:</b> An important part of RhyzUp is our antitoxin-toxin based Killswitch. This part of our construct ensures the bacteria stays contained in the deliberately inoculated plant’s rhizosphere, as the survival of the bacteria is directly linked to its direct sensing of the plant roots.
- To learn the specifics of our Killswitch system and how we developed and tested it visit our <a href="{{ url_for('pages', page='engineering') }}" class="hyperlink-under">Engineering page</a>.</p>
+ To learn the specifics of our Killswitch system and how we developed and tested it, visit our <a href="{{ url_for('pages', page='engineering') }}" class="hyperlink-under">Engineering page</a>.</p>
<p><b><i>P. sp.</i> IsoF - a rhizosphere regulator:</b> Though known for its competitiveness and ability to protect crops against pathogenic microbes, partially through active killing, our bacteria has a positive environmental impact. As RhyzUp de-deseritfies soil, it aids in the re-stabilization of a healthy rhizobiome, potentially boosting microbial diversity. These benefits would be most prevalent when implemented in controlled, closed system fields, such as greenhouses, or soils with a poor microbial diversity.
- To learn more about our plans of implementation visit our<a href="{{ url_for('pages', page='entrepreneurship') }}" class="hyperlink-under"> Implementation and Entrepreneurship page</a>.</p>
+ To learn more about our plans of implementation visit our<a href="{{ url_for('pages', page='entrepreneurship') }}" class="hyperlink-under"> Entrepreneurship and Implementation page</a>.</p>
<p><b>Spread of antimicrobial resistances:</b> In order to stop the potential horizontal transfer of antibiotic resistance genes through our plasmid’s backbones, we plan to integrate all our parts in our chassi’s genome in the future.</p>
</div>
@@ -121,8 +121,8 @@
We ensured sterile conditions by working under bunsen burners and laminar flow hoods.
All surfaces were routinely decontaminated with 70% ethanol and hands were periodically washed.
All waste was autoclaved according to BSL2 regulations before being disposed of appropriately.
- Find more information on our safe lab techniques in our <a href="https://teams.igem.org/5250/safety/74a59fdc-35c7-4567-86fd-3bea339286b0" class="hyperlink-under">safety</a>
- on our <a href="https://teams.igem.org/5250/safety/74a59fdc-35c7-4567-86fd-3bea339286b0" class="hyperlink-under">Team page</a>.
+ Find more information on our safe lab techniques in our <a href="https://teams.igem.org/5250/safety/74a59fdc-35c7-4567-86fd-3bea339286b0" class="hyperlink-under">safety form</a>
+ on our <a href="https://teams.igem.org/5250" class="hyperlink-under">Team page</a>.
</p>
</div>
</div>
@@ -162,7 +162,7 @@
<div class="row mt-0">
<div class="col-lg-3 col-md-3 col-sm-12">
- <div class="contribution-box" style="height: 550px;">
+ <div class="contribution-box">
<h3>Written consent</h3>
<p>
We received written, informed consent of all interviewees to record the interviews and use the material appropriately.
@@ -172,7 +172,7 @@
</div>
</div>
<div class="col-lg-9 col-md-9 col-sm-12">
- <div class="contribution-box" style="height: 550px;">
+ <div class="contribution-box">
<h3>Political awareness</h3>
<div class="image-description">
<img src="https://static.igem.wiki/teams/5250/project/safety/s-3-min.jpg" alt="Discussion on the topic “designer babies†at our lab workshop for highschool students" class="img-fluid d-block mx-auto responsive-img-smaller">
@@ -183,7 +183,7 @@
<p>
As mentioned in Environmental safety, the use of GMOs and the study of synthetic biology are topics of political debate globally as well as locally in Switzerland.
We were cautious not to display any political opinions toward the matter. This applies especially to our wiki, lecture series, educational visits to schools and our lab workshop.
- To find out more about the educational aspect of our project check out our<a href="{{ url_for('pages', page='parts') }}" class="hyperlink-under"> Education page</a>.
+ To find out more about the educational aspect of our project check out our<a href="{{ url_for('pages', page='education') }}" class="hyperlink-under"> Education page</a>.
</p>
</div>
</div>