Difference between revisions of "Part:BBa K3280007"

Latest revision as of 02:16, 22 October 2019

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

This is a composite part. We call this part the MermAID circuit. 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 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.

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In order to verify if our part really workes, 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 DH5-the part with metal pickup chimera (Iara-) 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.

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 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 Difusion Disc 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 DH5alpha, 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 DH5alpha 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

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 3: / Line 3: @@
 <partinfo>BBa_K3280007 short</partinfo>
-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.
+This is a composite part. We call this part the MermAID circuit. 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.
 <h2>Characterization</h2>
@@ Line 10: / Line 9: @@
 ===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.
+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 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.
+[[File:T--USP_SaoCarlos-Brazil--MerR-Chimerainv.svg|700px|center|]]
-<h4>Growth Curve</h4>
-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.
+In order to verify if our part really workes, 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.
-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.]
+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.
-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.
+<h4>Growth Curve</h4>
+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 DH5-the part with metal pickup chimera (Iara-) 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.
+[[File:T--USP_SaoCarlos-Brazil--concentration0.jpeg|300px|]][[File:T--USP_SaoCarlos-Brazil--concentration7-5.jpeg|300px|]][[File:T--USP_SaoCarlos-Brazil--concentration20.jpeg|300px|]][[File:T--USP_SaoCarlos-Brazil--concentration200.jpeg|300px|]][[File:T--USP_SaoCarlos-Brazil--concentration2000.jpeg|300px|]]
+<p style="text-align: center">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.</p>
+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.
 <h4>Hg Difusion Disc Test</h4>
+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 <i>E. coli</i> DH5alpha, 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 DH5alpha culture was, or, in a indirect way, if the MermAID works or not.
 [[File:T--USP SaoCarlos-Brazil--difusion.jpg|700px|center|]]
+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.
+[[File:T--USP SaoCarlos-Brazil--difusiong1.png|500px|center|]]
+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.
+[[File:T--USP SaoCarlos-Brazil--difusiong2.png|500px|center|]]
+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.
+[[File:T--USP SaoCarlos-Brazil--difusiong3.png|500px|center]]
+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===