Difference between revisions of "Part:BBa K3280001"
 
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<h2>Characterization</h2>
 
<h2>Characterization</h2>
 
<h3>By Team iGEM19_USP_SaoCarlos-Brazil 2019</h3>
 
<h3>By Team iGEM19_USP_SaoCarlos-Brazil 2019</h3>
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We used this and others diguanylate cyclases (Yddv and YdeH) in pETSUMO plasmid to run the tests. We did experiments with and without agitation, and also testing the efficiency of biofilm adherence in coconut fiber. The quantification was made by optical absorption measurement of Crystal violet. To know the specific protocol we used, access [[https://2019.igem.org/Team:USP_SaoCarlos-Brazil/Experiments]].
 
  
 +
Cyclic di-GMP (c-di-GMP) is recognized as an intracellular signaling molecule that coordinates the “lifestyle transition” from motility to sessility and vice versa (i.e. dispersion). The correlation between high c-di-GMP concentration in the cell and biofilm formation or between low c-di-GMP levels and motility has been demonstrated in several bacterial species [1].
 +
 +
Our project include one protein that capture mercury and also produces biofilm to improve the fixation of the heavy metal. So one of our experiments was to characterize several cyclic diguanylate in order to choose the best one for our circuit through quantification of biofilm with crystal violet. To know the specific protocol we used, access https://2019.igem.org/Team:USP_SaoCarlos-Brazil/Experiments
 +
 +
[[File:T--USP_SaoCarlos-Brazil--DGCvector.png|500px|center]]
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This part is originated from Pseudomonas aeruginosa. In our project we used this Wspr to test biofilm quantification in order to see what diguanylate cyclases is more efficient in biofilm formation. We use this and others diguanylate cyclases (Yddv and YdeH) in pETSUMO plasmid to run the tests. We did experiments with and without agitation, and also testing the efficiency of biofilm adherence in coconut fiber.
  
 
[[File:T--USP_SaoCarlos-Brazil--Parts-DGC24.png|500px|center|Figure 1: Each column is the mean value of a sample with 5 identical experiments made in the same 24-well plate, with the exception of the static SOC medium which had 1 of its results masked due to their dissonance from the rest, implying external contamination.
 
[[File:T--USP_SaoCarlos-Brazil--Parts-DGC24.png|500px|center|Figure 1: Each column is the mean value of a sample with 5 identical experiments made in the same 24-well plate, with the exception of the static SOC medium which had 1 of its results masked due to their dissonance from the rest, implying external contamination.
 
]]
 
]]
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As it can be seen from  figure 1, the bacteria transformed with WspR did not show a higher biofilm production in a non-static condition, which was coherent with other biofilm growth experiments made with E. coli.  
 
As it can be seen from  figure 1, the bacteria transformed with WspR did not show a higher biofilm production in a non-static condition, which was coherent with other biofilm growth experiments made with E. coli.  
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It’s interesting to note that a certain quantity of biofilm was expected for the control, pETSUMO without DGC insert, since our bacteria naturally produces low rates of biofilm.  
+
It’s interesting to note that a certain quantity of biofilm was expected for the control, pETSUMO without DGC insert, since our bacteria naturally produces low rates of biofilm.
  
  
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[[File:T--USP_SaoCarlos-Brazil--Parts-DGC24.png|500px|center|Figure 2: Once again, 5 replicates were made, with dissonant results masked]]
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[[File:T--USP_SaoCarlos-Brazil--Parts-DGC48.png|500px|center|Figure 2: Once again, 5 replicates were made, with dissonant results masked]]
  
 
The 48h static plate showed lower biofilm formation if compared to the 24h static plate. This could indicate a natural tendency for the E. coli to not maintain the small amount of biofilm that they produced under static conditions, since forming this matrix it’s a costly metabolic function.   
 
The 48h static plate showed lower biofilm formation if compared to the 24h static plate. This could indicate a natural tendency for the E. coli to not maintain the small amount of biofilm that they produced under static conditions, since forming this matrix it’s a costly metabolic function.   
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As for the 48h agitated plate, the absorption rate didn’t change significantly for the bacteria transformed with WspR, even though we can see a small increase in the absorption bar, which could indicate a preference for a longer period of incubation under agitated conditions. Also, the agitated WspR showed a very heterogeneous growth in the 24 hour plate. We can infer that this is a form of response from this DGC and that these bacterias would reach a higher level of absorbance if a longer incubation time was tested.  
 
As for the 48h agitated plate, the absorption rate didn’t change significantly for the bacteria transformed with WspR, even though we can see a small increase in the absorption bar, which could indicate a preference for a longer period of incubation under agitated conditions. Also, the agitated WspR showed a very heterogeneous growth in the 24 hour plate. We can infer that this is a form of response from this DGC and that these bacterias would reach a higher level of absorbance if a longer incubation time was tested.  
  
 +
We observed a substantial increase in biofilm formation for plates incubated under agitation, as well as for longer periods of time. We also noted that under static conditions, cells tend to considerably decrease biofilm production. Moreover, the DGC that showed the fastest response in the matter of biofilm production was YddV, which managed to saturate the acetic acid solution with biofilm in at least 24 hours. The second fastest response was from the DGC YdeH, reaching the maximum absorbance level in 48 hours. The agitated wells containing wspR manage to increase its biofilm production in the span of 24 hours, but did not reach the same high levels as YdeH and YddV, a longer timespan is necessary to evaluate wspR biofilm production.
  
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The same results can be presented in a different manner, In order to evaluate the changes happening from 24h to 48h, we plotted the change in absorption against time:
 
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[[File:T--USP_SaoCarlos-Brazil--Parts-DGCest.png|500px|center|Figure 3
 
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]]
 
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[[File:T--USP_SaoCarlos-Brazil--Parts-DGCagit.png|500px|center|Figure 4
 
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]]
 
  
 
In the end of our experiments, we came to the conclusion that Wspr is the less efficient diguanylate cyclase when compared to YdeH and YddV.  
 
In the end of our experiments, we came to the conclusion that Wspr is the less efficient diguanylate cyclase when compared to YdeH and YddV.  
  
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In order to test our project in a large scale, we tried to grow and quantify biofilm in coconut fiber.
 
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[[File:T--USP_SaoCarlos-Brazil--Parts-DGC24f.png|500px|center|Figure 5
 
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]]
 
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Comparing with previous experiments, there is a possibly of biofilm adhesion on coconut fiber given it’s decreased stained in the plate reading. We measured absorbance of crystal violet in both components: fiber and plate, to confirmate the distribution of biofilm growth. Our plan was use LB broth as a normalization of the experiment but after measures we realized that could possibly occurred a saturation on absorbance value that didn’t surpassed 3,3.
 
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[[File:T--USP_SaoCarlos-Brazil--Parts-DGC48f.png|500px|center|Figure 6
 
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]]
 
  
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We obtained a similar result for 72 hours well plate for the fibers and an increase of growth in the plate compared with 48 hours. Limit of absorbance remained the same, reinforcing the idea of a saturated solution of crystal violet, preventing our initial comparison plan.
 
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So with this experiment we tried to measure the concentration of biofilm with coconut fiber. But we noticed that our results had a methodological problem because although we realized that there was a biofilm, with 48h and 72h of growth we reached the acid acetic and violet crystal saturation and we couldn’t conclude how much biofilm grown up adhered.
 
  
 
<h2>References</h2>
 
<h2>References</h2>

Latest revision as of 02:26, 22 October 2019


WspR Diguanylate Cyclase

WspR. This part is a protein that has a domain that increases levels of c-di-GMP to induce biofilm formation.

Usage and Biology

This part is originated from Pseudomonas aeruginosa. In our project we used this Wspr to test biofilm quantification in order to see what diguanylate cyclase is more efficient in biofilm formation. We use this and others diguanylate cyclases (Yddv and YdeH) in pETSUMO plasmid to run the tests. We did experiments with and without agitation, and also testing the efficiency of biofilm adherence in coconut fiber.

Characterization

By Team iGEM19_USP_SaoCarlos-Brazil 2019

Cyclic di-GMP (c-di-GMP) is recognized as an intracellular signaling molecule that coordinates the “lifestyle transition” from motility to sessility and vice versa (i.e. dispersion). The correlation between high c-di-GMP concentration in the cell and biofilm formation or between low c-di-GMP levels and motility has been demonstrated in several bacterial species [1].

Our project include one protein that capture mercury and also produces biofilm to improve the fixation of the heavy metal. So one of our experiments was to characterize several cyclic diguanylate in order to choose the best one for our circuit through quantification of biofilm with crystal violet. To know the specific protocol we used, access https://2019.igem.org/Team:USP_SaoCarlos-Brazil/Experiments

T--USP SaoCarlos-Brazil--DGCvector.png

This part is originated from Pseudomonas aeruginosa. In our project we used this Wspr to test biofilm quantification in order to see what diguanylate cyclases is more efficient in biofilm formation. We use this and others diguanylate cyclases (Yddv and YdeH) in pETSUMO plasmid to run the tests. We did experiments with and without agitation, and also testing the efficiency of biofilm adherence in coconut fiber.

Figure 1: Each column is the mean value of a sample with 5 identical experiments made in the same 24-well plate, with the exception of the static SOC medium which had 1 of its results masked due to their dissonance from the rest, implying external contamination.

As it can be seen from figure 1, the bacteria transformed with WspR did not show a higher biofilm production in a non-static condition, which was coherent with other biofilm growth experiments made with E. coli. It’s interesting to note that a certain quantity of biofilm was expected for the control, pETSUMO without DGC insert, since our bacteria naturally produces low rates of biofilm.


Figure 2: Once again, 5 replicates were made, with dissonant results masked

The 48h static plate showed lower biofilm formation if compared to the 24h static plate. This could indicate a natural tendency for the E. coli to not maintain the small amount of biofilm that they produced under static conditions, since forming this matrix it’s a costly metabolic function. As for the 48h agitated plate, the absorption rate didn’t change significantly for the bacteria transformed with WspR, even though we can see a small increase in the absorption bar, which could indicate a preference for a longer period of incubation under agitated conditions. Also, the agitated WspR showed a very heterogeneous growth in the 24 hour plate. We can infer that this is a form of response from this DGC and that these bacterias would reach a higher level of absorbance if a longer incubation time was tested.

We observed a substantial increase in biofilm formation for plates incubated under agitation, as well as for longer periods of time. We also noted that under static conditions, cells tend to considerably decrease biofilm production. Moreover, the DGC that showed the fastest response in the matter of biofilm production was YddV, which managed to saturate the acetic acid solution with biofilm in at least 24 hours. The second fastest response was from the DGC YdeH, reaching the maximum absorbance level in 48 hours. The agitated wells containing wspR manage to increase its biofilm production in the span of 24 hours, but did not reach the same high levels as YdeH and YddV, a longer timespan is necessary to evaluate wspR biofilm production.


In the end of our experiments, we came to the conclusion that Wspr is the less efficient diguanylate cyclase when compared to YdeH and YddV.


References

[1]. Ha DG, O'Toole GA. c-di-GMP and its Effects on Biofilm Formation and Dispersion: a Pseudomonas Aeruginosa Review. Microbiol Spectr. 2015;3(2):10.1128/microbiolspec.MB-0003-2014. doi:10.1128/microbiolspec.MB-0003-2014

[2] Valentini M, Filloux A. Biofilms and Cyclic di-GMP (c-di-GMP) Signaling: Lessons from Pseudomonas aeruginosa and Other Bacteria. J Biol Chem. 2016;291(24):12547–12555. doi:10.1074/jbc.R115.711507



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 393
    Illegal PstI site found at 465
    Illegal PstI site found at 683
    Illegal PstI site found at 807
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 393
    Illegal PstI site found at 465
    Illegal PstI site found at 683
    Illegal PstI site found at 807
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 361
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 393
    Illegal PstI site found at 465
    Illegal PstI site found at 683
    Illegal PstI site found at 807
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 393
    Illegal PstI site found at 465
    Illegal PstI site found at 683
    Illegal PstI site found at 807
    Illegal NgoMIV site found at 306
    Illegal NgoMIV site found at 636
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 450
    Illegal SapI.rc site found at 273
    Illegal SapI.rc site found at 287