Difference between revisions of "Part:BBa K1127008"

Revision as of 14:41, 3 October 2013

Gold sensing device (PgolTS+LacZ+GolS)

This device is constructed to recognize gold (HAuCl4) in its environment. GolS is a transcriptional activator of the promoter P(golTS). Upon binding with the gold, it dimerizes and binds to the recognition site within the promoter. We also include LacZ alpha that is useful for quantification of the device activity via Beta-galactosidase assay. The mechanism is simplified and shown in Figure 1.

The promoter PgolTS has identical function to the part BBa_K310009 submitted by the team iGEM 2010 UIUC-Illinois [1]. However, our promoter is much cleaner as it does not contain non-promoter sequences which may interfere with transcription of the genes downstream. In addition, codon usage of our golS has been optimised specifically for E. coli to ensure efficient translation.

Introduction

Gold ions are toxic to most of the organisms and can cause serious environmental problems. The toxicity appears from interactions of gold ions with proteins and later on with the membrane causing the cells to lyse (1). Some of the microorganisms developed proteins that are sensitive to certain metal ions and can activated the expression of responsive elements. For example CueR from E. coli can bind to gold ions as well as copper ions (2). Due to lack of specificity this promoter is not very useful in synthetic biology. We decided to use part of the golT/S operon from Salmonella typhimurium, except for golT protein. This operon is regulated only by gold ions (3) therefore we took the promoter PgolT/S and transcription activator golS.

Characterization

To determine activity of the gold sensing device, beta-galactosidase assays were run in triplicates using PNPG as the substrate. In all experiments, untransformed E. coli strain DH5 alpha or strain BL21 were used as our negative control. The protocols are described in more detail in the Protocols section.

We demonstrated that E. coli strain DH5 alpha transformed with BBa_K1127008 can respond to the gold. In Figure 2a, relative activity of the enzyme beta-galactosidase increased significantly in the presence of golS and promoter P(golTS) (DH5 alpha_AB) but reduced to the basal level when golS was missing (DH5 alpha_A). The results are well supported by statistics - Kruskal-Wallis X^2= 20.75, df = 7, p-value < 0.01. This signifies the function of golS as a gold-dependent transcriptional activator.

In Figure 2b, E. coli strain BL21 has background expression of the enzyme. Strong enzyme activities were observed in the absence of lacZ alpha (BL21 and BL21_B), so that no differences were observed (Kruskal-Wallis X^2= 6.69, df = 3, p-value > 0.05). This suggests that there could be beta-galactosidase in the chromosome of this E. coli strain. Further research has confirmed our hypothesis. Beta-galactosidase (accession no. = C6ELN6) is present in BL21 according to the gene database on uniprot.org.

We determined kinetics of the device. DH5 alpha were exposed to different gold concentrations for 16 hours to reach steady states. We found that the response was non-linear with distinct phases - exponential phase (0.0uM - 2.5uM AuCl4), stationary phase (2.5uM - 10.0uM AuCl4) and logarithmic decline phase (not shown). The data were plotted in Figure3a with simulation of the fitted logistic model. Please visit our Modelling section for more detail. As seen in the plot, the device is very sensitive to the gold as low as 0.5uM. Later on, it became saturated from about 10uM onwards. The saturation could be caused by several factor, such as maximum promoter activity, expression and stability of the proteins and diffusion limit of the gold. In Figure 3b, we ran more assays on the subpart P(golTS)+LacZ alpha to confirm the function of golS. It is obvious that the promoter alone can't respond to the gold (Kruskal-Wallis X^2= 4.68, df = 6, p-value > 0.5).

E. coli strain DH5 alpha were transformed with the device or one of the subparts. They were streaked on the LB agar plates supplemented with chloramphenicol and Xgal. If LacZ alpha is expressed, the enzyme will degrade the substrate Xgal and turn colonies blue. In Figure 4, the colonies appear blue apart from those on the DH5 alpha+golS plate. This supports our hypothesis that P(golTS) is the minimal unit for the expression of LacZ alpha.

Figures

References

1. Goodman, C. M., McCusker, C. D., Yilmaz, T. & Rotello, V. M. Toxicity of Gold Nanoparticles Functionalized with Cationic and Anionic Side Chains. Bioconjugate Chem. 15, 897-900 (2004).

2. Stoyanpv, J. V. & Brown, N. L. The Escherichia coli copper-responsive copA promoter is activated by gold. J. Biol. Chem. 273, 1407-1410 (2003).

3. Wei, W. et al Engineering a gold-specific regulon for cell-based visual detection and recovery of gold. Chem. Sci. 3, 1780-1784 (2012). Sequence and Features

Assembly Compatibility:

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COMPATIBLE WITH RFC[10]
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COMPATIBLE WITH RFC[12]
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COMPATIBLE WITH RFC[21]
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COMPATIBLE WITH RFC[23]
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INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 701
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COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1127008 short</partinfo>
-This is the device we use in our project so that the cell could recognize the presence of gold in its environment.
+This device is constructed to recognize gold (HAuCl4) in its environment. GolS is a transcriptional activator of the promoter P(golTS). Upon binding with the gold, it dimerizes and binds to the recognition site within the promoter. We also include LacZ alpha that is useful for quantification of the device activity via Beta-galactosidase assay. The mechanism is simplified and shown in Figure 1.
-GolS protein acts as a positive transcriptional regulator on PgolTS once it binds to gold.
-Part was designed so that you can detect and quantify the activity of the promoter via Beta-galactosidase assay. For convenience the ORF of LacZ is covered by restriction sites of SacII and SanDI which allows the cell to respond to gold by expressing something to meet your needs.
 The promoter PgolTS has identical function to the part BBa_K310009 submitted by the team  iGEM 2010 UIUC-Illinois [https://parts.igem.org/Part:BBa_K310009]. However, our promoter is much cleaner as it does not contain non-promoter sequences which may interfere with transcription of the genes downstream. In addition, codon usage of our golS has been optimised specifically for E. coli to ensure efficient translation.
@@ Line 13: / Line 11: @@
 ==Characterization==
-The device has LacZ alpha which is useful for characterization. To determine activity of the part, beta-galactosidase assay was performed using PNPG as the substrate. In all experiments, untransformed E. coli strain DH5 alpha or strain BL21 were used as our negative control. The protocols are described in more detail on our wiki page [http://2013.igem.org/Team:York_UK/Notebook.html?page=protocols].
+To determine activity of the gold sensing device, beta-galactosidase assays were run in triplicates using PNPG as the substrate. In all experiments, untransformed E. coli strain DH5 alpha or strain BL21 were used as our negative control. The protocols are described in more detail in the Protocols section.
-The experiments showed that E. coli strain DH5 alpha transformed with the device can respond to gold (AuCl4). In Figure 1A, production of the enzyme increased significantly in the presence of the full construct (DH5 alpha_AB) but reduced to the basal level in the absence of golS (DH5 alpha_A). This signifies the function of golS as a gold-dependent transcriptional activator.
+We demonstrated that E. coli strain DH5 alpha transformed with BBa_K1127008 can respond to the gold. In Figure 2a, relative activity of the enzyme beta-galactosidase increased significantly in the presence of golS and promoter P(golTS) (DH5 alpha_AB) but reduced to the basal level when golS was missing (DH5 alpha_A). The results are well supported by statistics - Kruskal-Wallis X^2= 20.75, df = 7, p-value < 0.01. This signifies the function of golS as a gold-dependent transcriptional activator.
-In Figure 1B, E. coli strain BL21 has background expression of the enzyme. In contrast to the DH5 alpha (untransformed) and the DH5 alpha_B (transformed with golS), strong enzyme activities were observed in the absence of lacZ alpha - BL21 and BL21_B . This suggests that there may be beta-galactosidase in the chromosome of this E. coli strain.
+In Figure 2b, E. coli strain BL21 has background expression of the enzyme. Strong enzyme activities were observed in the absence of lacZ alpha (BL21 and BL21_B), so that no differences were observed (Kruskal-Wallis X^2= 6.69, df = 3, p-value > 0.05). This suggests that there could be beta-galactosidase in the chromosome of this E. coli strain. Further research has confirmed our hypothesis. Beta-galactosidase (accession no. = C6ELN6) is present in BL21 according to the gene database on uniprot.org.
-In order to determine kinetics of the device, DH5 alpha were exposed to different gold concentrations for 16 hours to reach steady state. The response was non-linear with distinct phases -  exponential phase (0.0uM - 2.5uM AuCl4), stationary phase (2.5uM - 10.0uM AuCl4) and logarithmic decline phase (not shown). According to the data, the device is very sensitive to the gold as low as 0.5uM; later on it became saturated from about 10uM onwards. This may be caused by intrinsic properties of the device. The data were fit to the nonlinear logistic model. In Figure 1C, simulated data were generated and plotted for comparison with the observations. For more information about modelling, please go to our wiki page [http://2013.igem.org/Team:York_UK/Notebook.html?page=modeling].
+We determined kinetics of the device. DH5 alpha were exposed to different gold concentrations for 16 hours to reach steady states. We found that the response was non-linear with distinct phases - exponential phase (0.0uM - 2.5uM AuCl4), stationary phase (2.5uM - 10.0uM AuCl4) and logarithmic decline phase (not shown). The data were plotted in Figure3a with simulation of the fitted logistic model. Please visit our Modelling section for more detail. As seen in the plot, the device is very sensitive to the gold as low as 0.5uM. Later on, it became saturated from about 10uM onwards. The saturation could be caused by several factor, such as maximum promoter activity, expression and stability of the proteins and diffusion limit of the gold. In Figure 3b,  we ran more assays on the subpart P(golTS)+LacZ alpha to confirm the function of golS. It is obvious that the promoter alone can't respond to the gold (Kruskal-Wallis X^2= 4.68, df = 6, p-value > 0.5).
-Figure 1D shows minimal activity of the promoter. What is more, it justifies the mathematical formulation of the model that there is no unknown process e.g. gold precipitation (between 0uM and 10uM AuCl4) otherwise the basal expression would be modified with increasing gold concentrations.
+E. coli strain DH5 alpha were transformed with the device or one of the subparts. They were streaked on the LB agar plates supplemented with chloramphenicol and Xgal. If LacZ alpha is expressed, the enzyme will degrade the substrate Xgal and turn colonies blue. In Figure 4, the colonies appear blue apart from those on the DH5 alpha+golS plate. This supports our hypothesis that P(golTS) is the minimal unit for the expression of LacZ alpha.
-In Figure 2, we show streak plates of the E. coli strain DH5 alpha transformed with the whole device or one of the subparts. The growing media contained Xgal which turns blue in the presence of beta-galactosidase. It supports  that PgolTS is the minimal unit for lacZ alpha expression.
 ==Figures==
-[[File:IGEM York 2013 Figure1.jpg|800px|thumb|center]]
+[[File:Gold_sensing_mechanism.jpg|800px|thumb|center]]
+[[File:Gold_sensing_strains.jpg|800px|thumb|center]]
-[[File:IGEM York 2013 Figure2.jpg.JPG|800px|thumb|center]]
+[[File:Gold_sensing_kinetics.jpg|800px|thumb|center]]
+[[File:Gold_sensing_plates.jpg|800px|thumb|center]]
 ==References==