Part:BBa_K3280001
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
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.
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.
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.
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:
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
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 361
- 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 450
Illegal SapI.rc site found at 273
Illegal SapI.rc site found at 287
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