Composite
MermAID

Part:BBa_K3280007

Designed by: Amanda Araujo Silva ; Raquel González Sterman ; Luana Corsi Antonio ; Juliana Naomi Yamauti Costa ; Nayla Naomi Kusimoto Takeuti ; Vinicius El Khalili Borsato   Group: iGEM19_USP_SaoCarlos-Brazil   (2019-10-16)
Revision as of 21:59, 21 October 2019 by Furuta (Talk | contribs)


MermAID (Mercury binding peptide + CBD anchor + HlyA + tDGC)

This is a composite part. We call this part an “Iara alpha*”. This Biobrick has a bidirectional promoter of MerR, a dCBD anchor, a lead binding peptide (MBP), a HlyA tag to secretion and a tDGC to induce biofilm formation.


Characterization

By Team iGEM19_USP_SaoCarlos-Brazil 2019

Usage and Biology

This part represent our MermAID systems, which contains our chimera and a diguanylate cyclase. This biobrick has a bidirecional promoter MerR + metal binding peptide that makes our MermAID catches mercury from the contaminate waters. And it also has a di-guanylate cyclase, a gene that contain a GGDEF domain responsible for induce production of biofilm, this biofilm will help to improve captation of mercury. With the HlyA, that is a tag to secretion our protein with mercury and the dCBD is an anchor to connect both into the biofilm. Therefore, if our circuit is properly working.

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Our work plan was basically based on combining two biobricks in order to capture mercury (BBa_k3280005 and BBa_k3280006), a double cellulose binding domain (dCBD), which consisted in two cellulose binding domains from Trichoderma reesei cellobiohydrolases completed with linkers (BBa_k3280006) and a protein domain that increases levels of c-di-GMP to induce biofilm formation (BBa_k3280000). We wanted to achieve a system that could capture mercury from contaminated samples and from that, induce biofilm. We also wanted the captured metal to stay attached to a substrate, which was coconut fiber, therefore the cellulose binding domain (BBa_k1321340).

In addition to our main parts, we also had to use other biobricks to complete the system, such as linkers (BBa_k243005), a terminator (BBa_B0015) and a signal peptide, HlyA, used to target proteins for secretion via the Type I secretion pathway of gram-negative bacteria (BBa_B0015).

In order to verify if our part really worked, we performed two different tests: growth curve and Hg difusion disc test. With these 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.

Growth Curve

In order to evaluate if the expression of our chimera 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 DH5-the metal pickup 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 ”g/ml. For the transformated strain we also made the experiment with high mercury concentrations such as 200 and 2000 ”g/ml. The results are shown in the figure 1 below.

T--USP SaoCarlos-Brazil--groww0.jpgT--USP SaoCarlos-Brazil--groww7.5.jpgT--USP SaoCarlos-Brazil--groww20.jpgT--USP SaoCarlos-Brazil--groww200.jpgT--USP SaoCarlos-Brazil--groww2000.jpg

Figure 1: Growth curve of the bacteria expressing Iara-α and the unmodified strain in culture medium containing 0, 7.5, 20, 200 and 2000 ”g/ml of mercury.

As can be seen on the graphs above, in the experiment performed with 0 ”g/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 ”g/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.


Hg Difusion Disc Test

T--USP SaoCarlos-Brazil--difusion.jpg

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.


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
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1699
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2062
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1563


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