Difference between revisions of "Part:BBa K3280007"

Revision as of 19:55, 21 October 2019

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 chimera MermAID. 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.

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 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 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.

Hg Difusion Disc Test

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

@@ Line 19: / Line 19: @@
 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 figure 1 below.
-[[File:T--USP_SaoCarlos-Brazil--groww0.jpg|500px|]][[File:T--USP_SaoCarlos-Brazil--groww7.5.jpg|500px|]][[File:T--USP_SaoCarlos-Brazil--groww20.jpg|500px|]][[File:T--USP_SaoCarlos-Brazil--groww200.jpg|500px|]][[File:T--USP_SaoCarlos-Brazil--groww2000.jpg|500px|] 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 µg/ml of mercury.]
+[[File:T--USP_SaoCarlos-Brazil--groww0.jpg|300px|]][[File:T--USP_SaoCarlos-Brazil--groww7.5.jpg|300px|]][[File:T--USP_SaoCarlos-Brazil--groww20.jpg|300px|]][[File:T--USP_SaoCarlos-Brazil--groww200.jpg|300px|]][[File:T--USP_SaoCarlos-Brazil--groww2000.jpg|300px| 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 µg/ml of mercury.]]
 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.