Team:USP SaoCarlos-Brazil/Contribution
CHARACTERIZATION
DIGUANYLATE CYCLASES
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 our Protocols
.
We used these three DGCs 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.
REGISTRY |
TYPE |
DESCRIPTION |
TEAM |
LENGHT |
|---|---|---|---|---|
| BBa_K748003 | Coding | yddV Diguanylate Cyclase | iGEM12 HIT-Harbin | 1386 |
| BBa_K1019004 | Coding | YdeH Diguanylate Cyclase | iGEM13 Toronto | 891 |
| BBa_K3280001 | DNA | WspR Diguanylate Cyclase | iGEM2019 USP_SaoCarlos-Brazil | 994 |
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. 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 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.
MERCURY COLLECTION AND RESISTANCE
REGISTRY |
TYPE |
DESCRIPTION |
TEAM |
LENGHT |
|---|---|---|---|---|
| BBa_K3280007 | Composite | MermAID: Mercury binding peptide + CBD anchor + HlyA + tDGC | iGEM2019 USP_SaoCarlos-Brazil | 2355 |
This part represents the MermAID circuit. The biobrick has a bidirectional promoter MerR + metal binding peptide that induces our MermAID to attach to mercury from contaminated waters. It has a di-guanylate cyclase (BBa_K3280000), a gene that contain a GGDEF domain responsible for the induction of biofilm production, which improves captation of mercury. Also have HlyA (BBa_K554002), a tag to secrete our protein - mercury complex (and is from Secretion System Type I) and the dCBD, an anchor to connect both into the biofilm.
In order to verify if our part really works, we performed two different tests: growth curve and Hg disc diffusion test. With these we hoped to demonstrate that cells containing our biological circuit were more fit to survive in a medium containing Hg, and therefore prove that not only our protein was being expressed, but also that it was properly exerting its function.
One important point of our tests is that our strain don’t have all the proteins of Secretion System Type I that recognizes HlyA signal, so we should co-express the secretion machinery proteins. Therefore, our experiments didn’t contain a plasmid with these proteins, but even without them, our part showed to work in the presence of Hg.
Growth Curve
In order to evaluate if the expression of our protein confers resistance to mercury and determining which are the most appropriate mercury concentrations to the other experiments, we made growth curves of the transformed bacteria in culture medium containing different concentrations of mercury and compare with the results obtained for the unmodified strain.
For the experiment, we transformed into E. Coli DH5α the part with metal pickup chimera (Iaraα) and the GGDEF domain-containing protein (Q9X2A8) (MermAID), both regulated by the same Mer promoter. The insert was into the pBS1K3 vector.
We started the test with a low optical density and keep measuring it every 30 minutes within 8 hours (for the transformed bacteria) and within 6 hours (for the unmodified strain). The unmodified strain was growth in culture medium Luria-Bertani (LB) containing different concentrations of mercury such as 0, 7.5, and 20 µg/ml. For the transformated strain we also made the experiment with high mercury concentrations such as 200 and 2000 µg/ml. The results are shown in the figure 1 below.
As can be seen on the graphs above, in the experiment performed with 0 µg/ml the insert-containing bacteria had a slower growth compared to the unmodified strain. This behavior may be due to increased metabolic expenses of transformed bacteria to express the synthetic proteins. Moreover, the transformed bacteria was able to grow in culture medium containing 7.5 µg/ml while the unmodified strain failed to grow in this concentration. Thus we can infer that our metal pickup chimera, Iara-α, gave to the bacteria a greater resistance to mercury, making it able to survive in environments with the concentration of Hg tested. Also, this is an evidence that Iara-α is been expressed and it is working as desired. In higher concentrations neither the engineered bacteria nor the unmodified one survived, since mercury ions are highly toxic and this result was expected.
Hg Disc Diffusion Test
This test intends to assess the survival capacity of our transformed bacteria containing the insert MermAID. We expect that, due to it’s Iara-alpha expression and consequent binding to Hg, the bacteria would be able to survive more in a mercury contaminated environment than a non-transformed one. With this data, our bacterial growth shows up higher than a wild type E. coli DH5α, a consistent explanation would rely in the functionality of our biosynthetic circuit, allowing inference about its effectiveness.
The growth was to be compared through the bacteria presence or not, around a diffusion disc with solution of the salt HgCl2. The halo size and the proximity to the disc indicates how resistant the E. coli DH5α culture was, or, in a indirect way, if the MermAID works or not.
In order to properly analyse the relation between Hg presence and cell growth the halos were measured using ImageJ, to maximize measurements precision the area of each halo was characterized 5 times, we then took its average value and compared obtained values in each replicate. The next step is to plot histograms with the average values of our experiment replicates vs. the control culture, comparing cell behaviour around the Hg contaminated zones. We expect to find smaller halos, i.e. smaller radii of death zones, in transformed cells due to their capacity to capture Hg which is believed to enhance cells chance to survive in the hostile environment. The table below shows the measured radii for each plate and then the average values for each concentration.
The graph displayed above exhibits the constructed histogram of average radii vs. control culture. In this case, the culture was incubated for 12h and the results indicate that transformed cells were able to survive in Hg contaminated zones better than non transformed cells, in particular for higher Hg concentrations which cells were not expected to survive at all.
When comparing replicates behaviour, we are able to identify that transformed cells might have an optimal Hg concentration to outgrow non transformed cells around 1000 ug/mL. The results shown above indicate that the are cells are suitable for our projects interest due to the results obtained for concentrations above 20 000 ug/mL.
We measured the radius again after 24h. We can note by the graphic that the radius of transformed cells increased a little if compared to 12h, and the non-transformed cells didn’t show any difference between the previous measurement. It can indicate that our bacteria is more resistant in different concentrations of Hg, but they eventually reach a threshold point, and also start to die which, increases the radius. Although the radius had increased in 24h for transformed cells, it is still smaller than non-transformed cells, which can support our hypothesis that our circuit helped to improve mercury resistance.
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
