Difference between revisions of "Part:BBa K748003"

Revision as of 02:20, 22 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

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, YdeH) in pETSUMO plasmid to run the test. We did experiments with and without agitation, and we also tested the efficiency of biofilm growth 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 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, YdeH.

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

After 48h the absorption rate didn’t change significantly for the bacteria transformed with YddV, indicating a possible saturation in the 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.

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 YddV showed the the fastest biofilm production when incubated under agitation, which managed to saturate the acetic acid solution with biofilm in at least 24 hours. 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]

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 After 48h the absorption rate didn’t change significantly for the bacteria transformed with YddV, indicating a possible saturation in the 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.
-[[File:T--USP_SaoCarlos-Brazil--Parts-DGCest.png|500px|center|Figure 3
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-[[File:T--USP_SaoCarlos-Brazil--Parts-DGCagit.png|500px|center|Figure 4
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 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.