Team:USP SaoCarlos-Brazil/Results
RESULTS
BIOLOGICAL EXPERIMENTS
We started out planning a myriad of different circuits in order to accomplish our main goal: to remove heavy metals from contaminated waters. We got carried away by our inspiration! The magnitude of the disaster moved us and sparked our need to aim high in the project’s impact on community. We underestimated the time it would take to get all we had planned done and sadly weren’t able to accomplish all the proposed circuits in the time we had. Given that, for future steps we can finish our carefully planned designs and evolve to a real applicable biofilter, making it affordable and ecofriendly, focusing on giving back to the society.
The diagram below indicates which constructions of our plan we were able to achieve
DIGUANYLATE CYCLASES
Biofilm formation
Pilot biofilm test to determine the ideal growth conditions and best protocol parameters:
This graphic comproves our visual analyses, showing that YdeH DGC bar is higher than the others, and wspr lower. We then ran our official tests using the same protocol used in the pilot-test. To decrease bar error, we added one more replicate on the plate and different incubation times. We also added a plate to grow static, for future comparison between methods. We maintained 1 mM IPTG concentration for all wells.
1 |
2 |
3 |
4 |
5 |
6 |
|
|---|---|---|---|---|---|---|
| A | Blank | Control | ||||
| B | wspr | |||||
| C | YdeH | |||||
| D | yddv | |||||
Plates showed a clear difference to the naked eye, as the intense stained on the last line of the right plate on the image. Experiment with agitation has contamination on the blank, possibly due to movimentation of the culture medium inside incubator that could overflow from the well. Less homogeneity is also observed in the second plate, showing that experiment with agitation could provide more unstable conditions.
As it can be seen from graphic, all DGCs 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 was clearly YddV, with an absorption rate more than 6 times higher than the second biggest producer, agitated YdeH.
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 YddV and WspR, indicating a possible saturation in the amount of biofilm maintained by those cells at approximately 3 Abs. The cells transformed with YdeH 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 WspR showed a very heterogeneous growth similar to the agitated YdeH 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.
To conclude, 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.
Biofilm formation on coconut fiber
Our final system should include the presence of coconut fiber in the biofilter that works as substrate to adhesion of biofilm. This experiment has the idea to simulate the real situation and verify the percentage of biofilm on the fiber that was immersed in a culture of transformed bacterias.
Previously in our saga, we obtained that growth with agitation showed the best results and increase of time, also exhibited increase of biofilm production. Therefore, we planned 24 well plates for 48 and 72 hours with agitation following quantification of coconut fiber biofilm including an extra part for the coconut fiber.
We used a coconut fiber that was characterized by the quantification fiber production So to put in the 24 well plate we did little balls of them with weight 60 mg.
- pETSUMO::YdeH (BBa_K1019004) - YdeH
- pETSUMO:: wspr (BBa_K3280001) - wspr
- pETSUMO:: yddv (BBa_K748003) - YddV
- pETSUMO::empty - Control
- Blank - SOC medium without bacteria
1 |
2 |
3 |
4 |
|
|---|---|---|---|---|
| A | Blank | Controle | ||
| B | wspr | |||
| C | YdeH | |||
| D | yddv | |||
After 48h and 72h we separated the fiber and put in another plate (let’s call plate F). After the part using violet crystal in our plates we could see qualitatively some differences between the amount of biofilm in the plate and in the fiber. On the image below we can notice an intense color of crystal violet on the wells by naked eyes, probably due the high absorption of the fiber:
Analysing after addiction acid acetic to our plate we noticed that some samples of control and blanks were stained as violet as the DGCs, it probably is a sign for contamination between the wells.
Once we finished preparing the plates we measured them by absorbance with 590nm.
Comparing with previous experiments, there is a possibly of biofilm adhesion on coconut fiber given it’s decreased stained in the plate reading. We measured absorbance of crystal violet in both components: fiber and plate, to confirmate the distribution of biofilm growth. Our plan was use LB broth as a normalization of the experiment but after measures we realized that could possibly occurred a saturation on absorbance value that didn’t surpassed 3,3.
We obtained a similar result for 72 hours well plate for the fibers and an increase of growth in the plate compared with 48 hours. Limit of absorbance remained the same, reinforcing the idea of a saturated solution of crystal violet, preventing our initial comparison plan.
So with this experiment we tried to measure the concentration of biofilm with coconut fiber. But we noticed that our results had a methodological problem because although we realized that there was a biofilm, with 48h and 72h of growth we reached the acid acetic and violet crystal saturation and we couldn’t conclude how much biofilm grown up adhered.
METAL COLLECTION AND RESISTANCE
Hg disc diffusion test
We expect that due to it’s Iara-alpha expression and consequently binding to Hg, the bacteria would be able to survive more in a mercury stressful environment rather than a non-transformed one. With this data, we may assure that our bacteria grows more than the wild type E. coli DH5alpha, a consistent explanation would rely in the functionality of our biosynthetic circuit, allowing inference about its effectiveness.
The halo size and the proximity to the disc indicates how resistant the E. coli DH5alpha culture was, or, in a indirect way, if the MermAID works or not.
Look to the plate results, was observed that non transformed E. coli DH5alpha, our biosynthetic circuit functionality control, grew in all 3 Hg concentrations, which wasn’t expected, since they do not have the insert that would confer them resistance to Hg, which is toxic and theoretically causes its death. Another aspect observed was that every plate with 20 mg/mL Hg concentration presented a halo zone of the same size, while in the others lower concentrations the bacterial culture spread through all plate. It means that for some reason our bacteria performed just like a non transformed one, having the same Hg radius of inhibition.
The plates analysis lead us to the formulation of 3 hypothesis to explain what happened:
- The Hg quantity applied in each filter paper circle, 10 uL, wasn’t enough to cover the hole plate;
- The 200 ug/mL and 20 ug/mL Hg concentrations are small and allow non transformed bacterias to survive;
- The bacteria does not contain our insert.
In front of this results and hypothesis, we decided to repeat the experiment, changing some parameters. We tested the size of the filter paper circle, to guarantee that the volume used was enough to wet, but not to soak it. With this, we decided that the initial size was perfect. Also applied Hg solution in a more systematic way to guarantee the same waiting time and to establish a more solid replicable method.
In the following attempt, to ensure formation of different halo zones sizes, we added three different Hg concentrations to the previous ones: 2000 ug/mL, 10000 ug/mL, 40000 ug/mL and excluded the lower one of 20 ug/mL. This way, we could analyse if there’s a minimum Hg concentration that belows it the natural cells can survive without trouble and beyond our circuit is necessary for bacterial survival. The last variable we changed was the colony used, the A3. More details of why can be found in Lab Notes, date 10.19.2019.
The plates were made in triplicate and each large plate was divided in six, so that every plates had all Hg concentrations. The indicated control is the non transformed E. coli DH5alpha:
Hg disc diffusion test plates, with concentrations ranging from zero to 40 mg/uL. 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 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.
Graphic: Measurements of halos from mercury stained colonies after 24h. 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. With the images below we can objectvely see what was discussed above, explicited with the Hg halo area comparison:
Growth curve
This experiment consists in mapping the bacterial growth of E. coli DH5alpha containing our biosynthetic circuit versus non transformed ones, in medium with different mercury concentrations. This way, we would be able to compare the behaviour of our transformed cells in the presence of Hg, with non transformed ones. With this we hoped to demonstrate that cells containing our biological circuit were more fit to survive in and medium containing Hg, and therefore prove that not only our protein was being expressed, but also that it was properly exerting its function to capture mercury.
We worked with some of the previous Hg concentrations used in Hg diffusion disc test and another ones between (0-200) ug/mL: 0 ug/mL, 7.5 ug/mL (same concentration of team UFAM 2016 used), 20 ug/mL, 200 ug/mL, 2000 ug/mL. Cells were incubated at 37°C under agitation. The measurements were made every 30 minutes starting with OD600 = 0,05.
For the experiment, we transformed into E. Coli DH5- the metal binding chimera (Iara-) and the GGDEF domain-containing protein (Q9X2A8), both regulated by 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 bacteria 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 images 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 bactéria 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 this result was expected.
These experiments also showed that our MermAID works even without the Secretion System, which would be responsible to lead our protein out of the cell. Instead of binding to our protein outside the cell, the mercury penetrates the cell, where our protein binds to it. The downside is that bioaccumulation leads to cell death for high mercury concentrations.
This behaviour is evidenced in the growth curves, because even though transformed cells survived longer for low concentrations, when compared to the non transformed ones, as we increased the Hg concentration, they could have reached a threshold, saturating the amount of protein available to capture the mercury, and consequently causing cell death.
HARDWARE EXPERIMENTS
COCONUT FIBER
Adsorption of water
The coconut fiber water absorption test was performed so that we could better plan the volume of fiber and the water flow we should be using in the filter.
We weighed 1g of coconut fiber, which was placed in 50mL beakers and we added different volumes of distilled water until complete material humidification (overnight), which was determined by visual observation if there is visible water in aqueous phase.
The table below shows the volumes used and whether or not it has been absorbed.
Volume of water (mL) |
Absorbed? |
|---|---|
| 2.5 | yes |
| 3.5 | no |
| 5.0 | no |
| 10.0 | no |
| 20.0 | no |
As can be seen from the table, of all tested volumes only 2.5mL was fully absorbed by the fiber after the analyzed period. Therefore, we know that the absorption of our fiber is 2.5mL/g.
Fourier-transform infrared spectroscopy
The FTIR equipment was used as part of the characterization of our fiber since it was possible to determine the presence of cellulose in our coconut fiber. This is important because as our mercury-collecting chimera has a cellulose-binding tag (inserted by us into the genetic circuit), the protein can bind to both the bacterial biofilm matrix and coconut fiber (if it had a significant percentage of cellulose).
We used the FTIR equipment from the laboratory of the Nanotechnology and Nanotoxicology Group (GNano) of our Institute. With the help of postdoctoral student Bianca Martins Estevão, we made KBr pellets with the green coconut fiber powder, and with these pellets, we were able to collect the FTIR spectrum of our fiber.
Comparative analysis of the spectrum with the literature shows that there are bands attributed to hydroxyl groups (O-H cellulosic stretch) at 3430 cm-1, axial deformation of C-H groups at 2910 cm-1, angular deformation of C-H groups 1375 cm-1, angular deformation of primary alcohol C-O bonds at 1167 cm-1, C-O-C bond absorption band, representing pyranose ring vibration at 1050 cm-1 and β-glycosidic bonds between glycan units in 901 cm-1, characteristics of cellulose. The band at 1745 cm-1 refers to acetate group residues from hemicellulose.
With this result, we could conclude that coconut fiber could possibly serve as a good substrate for the cellulose binding anchor of our metal-collecting chimera.
MERCURY QUANTIFICATION
MERCURY CONCENTRATION
Due to the high cost of mercury concentration measurements and the need of relatively high volumes of mercury solutions that would be required for laboratories to provide reasonable results using traditional methods of analysis, as ICP-OES [1], our team had to work on alternative methods of metal quantification or even semi-quantitative analysis procedures.
Thus, we contacted Renan Romano, PhD student at IFSC-USP, and elaborated a semi-quantitative approach using Laser Induced-Breakdown Spectroscopy (LIBS). The available equipment to carry on this analysis belongs to Empresa Brasileira de Pesquisa Agropecuária (Embrapa), São Carlos unit [2].
LASER INDUCED-BREAKDOWN SPECTROSCOPY (LIBS)
Laser induced-breakdown spectroscopy, LIBS, is an analytical technique that allows identifying the elemental composition of solid, liquid and gaseous samples, returning clean and precise data. The technique is almost non-destructive due to the small areas that are in fact ablated by the laser beam, corresponding to nano to micrograms of sample mass [4].
The procedure consists in a high energy pulsed laser that hits the sample directly causing solid samples, in particular, to melt and vaporize. After the vaporization, the material is excited to a higher energy level, when the sample decays it emits radiation in element-specific wavelengths, thus it is possible to identify different compounds [4]. Figure 1 below depicts the system’s main components.
MERCURY ANALYSIS ESSAY BY LIBS
Renan has put us in contact with another PhD student, Alfredo Augusto Xavier, who works in Optics and Photonics Lab at Embrapa Instrumentation section. The department's supervisor, Dra Débora Milori, has allowed us to use the LIBS equipment.
Alfredo, then, not only taught how to use the equipment set up but also help us in sample preparation. To perform measurements in aqueous solution, KBr pellets were used.
Using several mercury concentrations in solutions we performed the calibration. We then dripped 10 uL of our solution on different KBr pellets and left it drying for a few minutes before putting it to the sample holder. This procedure was repeated for all solutions.
LIBS RESULTS PROCESSING
When processing the results, Renan once again helped us out. For every laser hit, a spectral emission curve was generated, and after 100 laser hits, we were able to generate an average emission spectrum of samples.
We have previously searched for Hg emission peak wavelength, found it at 194.15 nm and 253.65 nm, thus when treating the collected data we could analyze with higher precision the regions around these peaks, proceeding to data normalization right after.
Finally, we plotted the graphs shown below, they show the emission spectrum and emission bar graphs of both of the Hg emission peaks.
RESULTS
The KBr powder to make the KBr pellet was 98% pure and there was a low concentration of Fe II. Fe II has a characteristic emission peak that is very close to one of the mercury emission peaks at 253.65nm.
When analyzing the emission spectrum of KBr pellets with and without mercury solution, we found no significant difference in the peak relative to the emission of 253.65nm, probably due to Fe II emission present in the KBr pellet composition. Thus, we chose to take into account only the part of the spectrum referring to the 194.15nm peak.
Graph 1 shows the average emission peak of 194.15nm for each of the samples analyzed. In it, it can be noted that there is no pattern of increased intensity of mercury emission due to the increased concentration of the solution deposited in the pellet.
Graph 2 represents the total average peak area at 194.15nm for each of the samples. As in graph 1, there is no increase in mercury emission due to the increase in metal concentration.
In both cases, it is possible to observe that although the concentrations were increasing, the peak did not have an increasing behavior for all samples, probably due to the inhomogeneous distribution of the solution in the tablet because its surface was not completely flat, causing the solution to distributed differently in each pellet. Moreover, the concentration spectra that would be used in our experiments do not have a significant intensity to evaluate the peak.
Therefore, as we did not obtain a conclusive result to relate peak intensity to sample concentration and we could not detect the mercury peak in lower concentration samples, we chose not to use the LIBS method to quantify our results.
REFERENCES
- [1] CADORE, S.; MATOSO, E.; SANTOS, M. C. A ESPECTROMETRIA ATÔMICA E A DETERMINAÇÃO DE ELEMENTOS METÁLICOS EM MATERIAL POLIMÉRICO. Quim. Nova, Vol. 31, No. 6, 1533-1542, 2008
- [2] Embrapa Instrumentações : https://www.embrapa.br/instrumentacao
- [3] COSTA, V. C.; AUGUSTO, A. S.; CASTRO, J. P. MACHADO, R. C.; ANDRADE, D. F.; BABOS, D. V.; SPERANÇA, M. A.; GAMELA, R. R.; PEREIRA-FILHO, E. R.
- LASER INDUCED-BREAKDOWN SPECTROSCOPY (LIBS): HISTÓRICO, FUNDAMENTOS, APLICAÇÕES E POTENCIALIDADES. Quim. Nova, Vol. 42, No. 5, 527-545, 2019.
- [4] CARLOS R. MENEGATTI, 1 GUSTAVO NICOLODELLI, 2 GIORGIO S. SENESI, 3 OTAVIO A. DA SILVA, 1 HÉLCIO J. I. FILHO, 1 PAULINO R. VILLAS BOAS, 2 BRUNO S. MARANGONI, 4 AND DÉBORA M. B. P. MILORI2,*Semiquantitative analysis of mercury in landfill leachates using double-pulse laser-induced breakdown spectroscopy. Vol. 56, No. 13 / May 1 2017 / Applied Optics.
