Difference between revisions of "Part:BBa K748003"
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yddV is a Diguanylate Cyclase-Genomic. The product of gene yddV has diguanylate cyclase (DGC) activity. DGC uses 2 GTP to form a Bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP). C-di-GMP is a global second messenger in bacteria. Biofilm formation of E.coli is manipulable by varying c-di-GMP concentrations. When the c-di-GMP level stays low, there is little biofilm and the bacteria is dispersive. High concentrations of c-di-GMP promote bacteria to form more cellulose and fimbriae, which enhances the biofilm formation and decrease the motility of bacteria.  
 
yddV is a Diguanylate Cyclase-Genomic. The product of gene yddV has diguanylate cyclase (DGC) activity. DGC uses 2 GTP to form a Bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP). C-di-GMP is a global second messenger in bacteria. Biofilm formation of E.coli is manipulable by varying c-di-GMP concentrations. When the c-di-GMP level stays low, there is little biofilm and the bacteria is dispersive. High concentrations of c-di-GMP promote bacteria to form more cellulose and fimbriae, which enhances the biofilm formation and decrease the motility of bacteria.  
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<h2>Characterization</h2>
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<h3>By Team iGEM19_USP_SaoCarlos-Brazil 2019</h3>
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===Usage and Biology===
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We used this and others diguanylate cyclases (Wspr 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.
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[[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 YddV showed a higher biofilm production in a non-static condition, with the exception of the bacteria transformed with WspR, which is coherent with other biofilm growth experiments made with E. coli. The DGC that produced higher amounts of biofilm under agitation in 24h was clearly YddV, with an absorption rate more than 6 times higher than the second biggest producer, agitated YdeH.
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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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After 48h we had a difference in the agitated plate for YdeH, the absorption rate didn’t change significantly for the bacteria transformed with YddV, indicating a saturation in the possible amount of biofilm maintained by those cells at approximately 3 Abs. 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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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 YddV showed the the fastest biofilm production, when incubated under agitation. Of all DGCs we tested, this was the more efficient one. 
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===References===
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[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
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[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
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 19:28, 21 October 2019

yddV is a Diguanylate Cyclase-Genomic.

yddV is a Diguanylate Cyclase-Genomic. The product of gene yddV has diguanylate cyclase (DGC) activity. DGC uses 2 GTP to form a Bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP). C-di-GMP is a global second messenger in bacteria. Biofilm formation of E.coli is manipulable by varying c-di-GMP concentrations. When the c-di-GMP level stays low, there is little biofilm and the bacteria is dispersive. High concentrations of c-di-GMP promote bacteria to form more cellulose and fimbriae, which enhances the biofilm formation and decrease the motility of bacteria.


Characterization

By Team iGEM19_USP_SaoCarlos-Brazil 2019

Usage and Biology

We used this and others diguanylate cyclases (Wspr 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.

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 YddV showed a higher biofilm production in a non-static condition, with the exception of the bacteria transformed with WspR, which is coherent with other biofilm growth experiments made with E. coli. The DGC that produced higher amounts of biofilm under agitation in 24h was clearly YddV, with an absorption rate more than 6 times higher than the second biggest producer, agitated YdeH.


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

After 48h we had a difference in the agitated plate for YdeH, the absorption rate didn’t change significantly for the bacteria transformed with YddV, indicating a saturation in the possible amount of biofilm maintained by those cells at approximately 3 Abs. 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.

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:

Figure 3


Figure 4

In the end of our experiments, we came to the conclusion that YddV showed the the fastest biofilm production, when incubated under agitation. Of all DGCs we tested, this was the more efficient one.


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
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 37
  • 1000
    COMPATIBLE WITH RFC[1000]