Difference between revisions of "Part:BBa K1019004"
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| − | [[File:T--USP_SaoCarlos-Brazil--Parts- | + | [[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. | ||
Latest revision as of 02:25, 22 October 2019
YdeH Diguanylate Cyclase
YdeH is a diguanylate cyclase that is involved in producing the key secondary messenger c-di-GMP. c-di-GMP is proposed to be a "master regulator" of biofilm formation.
Its expression increases surface adhesion and various aspects of the biofilm response.
For further details and additional references please visit [http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG11643 EcoCyc]
Characterization
By Team iGEM19_USP_SaoCarlos-Brazil 2019
Usage and Biology
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
We used this and others diguanylate cyclases (Wspr, YddV) in pETSUMO plasmid to run the test. We did experiments with and without agitation, and also testing the efficiency of biofilm in coconut fiber.
As it can be seen from figure 1, the bacteria transformed with YdeH showed a higher biofilm production in a non-static condition. This DGC did not produce significant amounts of biofilm, even though we can see a small increase in the absorption bar when under agitation. 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 changed significantly for the bacteria transformed with YdeH, and showed the biggest change in the absorption rate from 24 hours to 48 hours, indicating a preference for a longer period of incubation under agitated conditions.
The agitated YdeH showed a very heterogeneous growth, so 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 YdeH is very efficient in biofilm production when incubated for a minimum of 48h in agitation, presenting the second fastest response.
References
Escherichia coli K-12 substr. MG1655 Enzyme: diguanylate cyclase. (2013). Ecocyc. Retrieved May 20, 2013, from http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG11643
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
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 775
Illegal SapI.rc site found at 800