Difference between revisions of "Part:BBa K1355001:Experience"

Latest revision as of 01:35, 28 October 2016

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Mer Operon regulation system improviment to increase Hg bioremediation

Our team (UFAM-UEA_Brazil) worked improving the Mer operon expression to increase bioremediation in E. coli through novel mer promoters sequences. For it, we primarily characterized the MerR expression under control of different promoters from Anderson Collection (BBa_J23100, BBa_J23104, BBa_J23106 e BBa_I142033) through the repression of RFP (BBa_K081014) production, in a synthetic genetic circuit represented bellow.

Figure 02: MerR expression test.

On the first experiment, we used the novel regulated promoter designed by our team BBa_K2123109 (Stationary growth phase promoter with downstream mer operator) to measure the RFP repression by MerR under control of BBa_J23100, BBa_J23104, BBa_J23106 e BBa_I142033 constitutive promoters in solid LB media, as you can see below.

Figure 03: MerR repressing RFP production in different levels cloned in E. coli DH5-alpha.

You can analyse by the RFP expression and thus fluorescence intensity that the samples greater repressed by MerR was the ones under control of BBa_J23100 and BBa_I14033 constituve promoters. So, we measured the RFP expression using Chamaleon Spectrofluorometer with and without MerR repressor protein under control of these two constituve promoter. The results are presented in the graph 1 below.

Graph 1: RFP expression with and without MerR regulator under control of BBa_J23100 and BBa_I14033. Control bacteria is DH5-alpha without any plasmid vector.

As we can see, between BBa_J23100 and BBa_I14033, the best repressed was with BBa_J23100 constitutive promoter. So, we selected BBa_J23100 to controls MerR expression in our synthetic genetic circuits. We made also other experiments to understand the interactions between MerR and new regulated promoters designed by our team, aiming to reach a well repressor mechanism, increasing the natural MerR regulation. In this way, we measured MerR regulation with two more regulated promoters: BBa_K2123102 and BBa_K2123101 - Tac promoter + overlapped and between mer operator, respectively expression RFP. The MerR regulation mechanism was characterized by repressing RFP expression and therefore reducing fluorescence intensity according to the regulator efficiency. We measured it also utilizing Chamaleon Spectrofluorometer. The results are presented in the graph 2 and 3 below.

Graph 02 and 03: RFP expression with and without MerR regulator under control of BBa_J23100 constitutive promoter, repressing BBa_K2123101 and BBa_K2123102 new regulated promoters. We achieved almost a totally repression with this BioBrick part! Awesome results, as you can see in the figures below.

Figure 04: RFP expression regulation by MerR.

To finalize this part characterization, we cloned it with the Mer Operon improved genes device, as the figure below, aiming to measure the amoung of mercury bioremediated under control of our MerR with very strong regulation in 7.5ppm of mercury chloride with 10 hours of growth.

Previous iGEM projects achieved 75% of mercury bioremediated with their synthetic genetic circuits. With our improvement, designing new regulated promoters sequences and thus increasing MerR regulation, this amoung increased to =97%= of mercury bioremediation in E. coli DH5-alpha. We measured with DMA-80 (Direct Mercury Analyser) equipment. Check this out below!

Graph 04: Comparison growth curve of previous synthetic genetic circuits (BBa_K1355004) with our improved devices in 7.5ppm of mercury chloride, measured with spectrophotometer (600nm wavelenght).

Graph 05: Amoung of mercury after 10 hours of bacterial growth with our construction (BBa_K2123108). So, to validate it, our team constructed the first real bioreactor for mercury bioremediation of iGEM! See the results below!

After 18h, our construction reached 70% of mercury bioremediation! Want to see more? Access our wiki: 2016.igem.org/Team:UFAM-UEA_Brazil.

User Reviews

UNIQ07ebe5f5ff47ad46-partinfo-00000000-QINU UNIQ07ebe5f5ff47ad46-partinfo-00000001-QINU

@@ Line 4: / Line 4: @@
 how you used this part and how it worked out.
-===Applications of BBa_K1355001===
+===Mer Operon regulation system improviment to increase Hg bioremediation===
-'''Experiments and Results as a Biosensor (BBa_K1355001 + BBa_E0840)'''
+Our team (UFAM-UEA_Brazil) worked improving the Mer operon expression to increase bioremediation in E. coli through novel mer promoters sequences. For it, we primarily characterized the MerR expression under control of different promoters from Anderson Collection (BBa_J23100, BBa_J23104, BBa_J23106 e BBa_I142033) through the repression of RFP (BBa_K081014) production, in a synthetic genetic circuit represented bellow.
+<center>https://static.igem.org/mediawiki/parts/6/6a/UFAM_UEA_MERR_PART_2.png</center>
+<center><b>Figure 02:</b> MerR expression test. </center>
-The experiment to quantify GFP expression induced by Hg was made according to the protocol “Quantification of Green Fluorescent Protein (GFP) induced by different concentrations of mercury in Escherichia coli DH5α”.
+On the first experiment, we used the novel regulated promoter designed by our team BBa_K2123109 (Stationary growth phase promoter with downstream mer operator) to measure the RFP repression by MerR under control of BBa_J23100, BBa_J23104, BBa_J23106 e BBa_I142033 constitutive promoters in solid LB media, as you can see below.
-DH5α transformed with BBa_K1355002 was inoculated in LM (LB with low concentration of NaCl) liquid medium with chloramphenicol and grew until the Optical Density was 0.4 to 0.6abs (measured on spectrophotometer at 600 nm wavelength). After cell growth, an aliquot of 500μl in 5 eppendorf tubes (2ml) was taken and then added mercury chloride in order to achieve the concentrations of: 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml. The samples were incubated at 37°C on shaker. We collected each eppendorf tube at time 1 (01:30 hours of incubation), time 2 (03:00 hours of incubation) and time 3 (04:30 hours of incubation). Every sample was centrifuged at 12000g for 3 minutes and the pellet washed with TN Buffer (Nacl 0.15M + Tris HCl 10mM) and then re-suspended with 500μl of the same buffer. The same process was made to the bacterium without construction as a control to GFP expression/intensity. GFP expression was measured using the Hidex Chameleon spectrofluorimeter with excitation filter 340 nm and emission filter 500 nm wavelength. The Optical Density was measured simultaneously. All samples were analyzed in triplicate.
-The graph represented on Figure 1 shows the Optical Density of transformed DH5α with BBa_K1355002 in different Hg concentrations in function of time:
+<center>https://static.igem.org/mediawiki/parts/b/bb/Imagem3.png</center>
-[[File:bs1.png]]
+<center><b>Figure 03:</b> MerR repressing RFP production in different levels cloned in E. coli DH5-alpha.</center>
-'''Figure 1.''' Optical Density measured in the four given times, at mercury chloride concentrations of 0 µg/ml, 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml.
+You can analyse by the RFP expression and thus fluorescence intensity that the samples greater repressed by MerR was the ones under control of BBa_J23100 and BBa_I14033 constituve promoters. So, we measured the RFP expression using Chamaleon Spectrofluorometer with and without MerR repressor protein under control of these two constituve promoter. The results are presented in the graph 1 below.
+<center>https://static.igem.org/mediawiki/parts/d/db/Ufam_uea_merr_part4.png</center>
-In these condition cell growth increases along time. The highest values correspond to bacteria not exposed to mercury or to small concentrations, as in 0 µg/ml, 0.01 µg/ml and 0.02 µg/ml. Suggesting a harmless condition to bacteria. However, cell growth decreases at higher concentrations as, 0.1 µg/ml, 0.2 µg/ml and especially 1µg/ml, giving to bacteria a hard time for development.
+<b>Graph 1:</b> RFP expression with and without MerR regulator under control of BBa_J23100 and BBa_I14033. Control bacteria is DH5-alpha without any plasmid vector.
-The graph represented on Figure 2 shows the fluorescence emitted by DH5-alpha induced by different Hg concentrations in function of the time; and the graph represented on Figure 3, shows the ratio between fluorescence emitted and Optical Density.
-[[File:bs2.png]]
+As we can see, between BBa_J23100 and BBa_I14033, the best repressed was with BBa_J23100 constitutive promoter. So, we selected BBa_J23100 to controls MerR expression in our synthetic genetic circuits. We made also other experiments to understand the interactions between MerR and new regulated promoters designed by our team, aiming to reach a well repressor mechanism, increasing the natural MerR regulation. In this way, we measured MerR regulation with two more regulated promoters: BBa_K2123102 and BBa_K2123101 - Tac promoter + overlapped and between mer operator, respectively expression RFP. The MerR regulation mechanism was characterized by repressing RFP expression and therefore reducing fluorescence intensity according to the regulator efficiency. We measured it also utilizing Chamaleon Spectrofluorometer. The results are presented in the graph 2 and 3 below.
-'''Figure 2.''' GFP fluorescence intensity in the four given times, at mercury chloride concentrations of 0 µg/ml, 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml.
+<center>https://static.igem.org/mediawiki/parts/f/f8/Ufam_uea_merr_part5.png</center>
-[[File:bs3.png]]
+<b>Graph 02 and 03:</b> RFP expression with and without MerR regulator under control of BBa_J23100 constitutive promoter, repressing BBa_K2123101 and BBa_K2123102 new regulated promoters.
+We achieved almost a totally repression with this BioBrick part! Awesome results, as you can see in the figures below.
-'''Figure 3.''' GFP fluorescence per cell growth ratio in the four given times, at mercury chloride concentrations of 0 µg/ml, 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml.
+<center>https://static.igem.org/mediawiki/parts/b/bd/Ufam_uea_merr_part6.png</center>
-Fluorescence can be observed in bacteria exposed to small concentrations, as 0.01µg/ml and 0.02µg/ml, but has low intensity. Fluorescence levels presents medium intensity in the higher concentration as consequence of cell death. Fluorescence intensity increased more than 480% in 0.2µg/ml concentrations, compared to fluorescence intensity from 1µg/ml, even in cell growth reduction, demonstrating its efficiency to induce mer promoter.
+<b>Figure 04:</b> RFP expression regulation by MerR.
-'''Experiments and Results as a Biosensor (BBa_K1355001 + BBa_E0840)'''
+To finalize this part characterization, we cloned it with the Mer Operon improved genes device, as the figure below, aiming to measure the amoung of mercury bioremediated under control of our MerR with very strong regulation in 7.5ppm of mercury chloride with 10 hours of growth.
-The experiment to quantify GFP expression induced by Hg was made according to the protocol “Quantification of Green Fluorescent Protein (GFP) induced by different concentrations of mercury in Escherichia coli DH5α”.
+<center>https://static.igem.org/mediawiki/parts/7/7e/UFAM_MERBA_10.png</center>
-DH5α transformed with BBa_K1355002 was inoculated in LM (LB with low concentration of NaCl) liquid medium with chloramphenicol and grew until the Optical Density was 0.4 to 0.6abs (measured on spectrophotometer at 600 nm wavelength). After cell growth, an aliquot of 500μl in 5 eppendorf tubes (2ml) was taken and then added mercury chloride in order to achieve the concentrations of: 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml. The samples were incubated at 37°C on shaker. We collected each eppendorf tube at time 1 (01:30 hours of incubation), time 2 (03:00 hours of incubation) and time 3 (04:30 hours of incubation). Every sample was centrifuged at 12000g for 3 minutes and the pellet washed with TN Buffer (Nacl 0.15M + Tris HCl 10mM) and then re-suspended with 500μl of the same buffer. The same process was made to the bacterium without construction as a control to GFP expression/intensity. GFP expression was measured using the Hidex Chameleon spectrofluorimeter with excitation filter 340 nm and emission filter 500 nm wavelength. The Optical Density was measured simultaneously. All samples were analyzed in triplicate.
-The graph represented on Figure 1 shows the Optical Density of transformed DH5α with BBa_K1355002 in different Hg concentrations in function of time:
+Previous iGEM projects achieved 75% of mercury bioremediated with their synthetic genetic circuits. With our improvement, designing new regulated promoters sequences and thus increasing MerR regulation, this amoung increased to =97%= of mercury bioremediation in E. coli DH5-alpha. We measured with DMA-80 (Direct Mercury Analyser) equipment. Check this out below!
-[[File:bs1.png]]
+<center>https://static.igem.org/mediawiki/parts/e/ef/Ufam_uea_merr_part7.png</center>
-'''Figure 1.''' Optical Density measured in the four given times, at mercury chloride concentrations of 0 µg/ml, 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml.
+<b>Graph 04:</b> Comparison growth curve of previous synthetic genetic circuits (BBa_K1355004) with our improved devices in 7.5ppm of mercury chloride, measured with spectrophotometer (600nm wavelenght).
+<center>https://static.igem.org/mediawiki/parts/a/ac/Ufam_uea_merr_part8.png</center>
+<b>Graph 05:</b> Amoung of mercury after 10 hours of bacterial growth with our construction (BBa_K2123108).
+So, to validate it, our team constructed the first real bioreactor for mercury bioremediation of iGEM! See the results below!
-In these condition cell growth increases along time. The highest values correspond to bacteria not exposed to mercury or to small concentrations, as in 0 µg/ml, 0.01 µg/ml and 0.02 µg/ml. Suggesting a harmless condition to bacteria. However, cell growth decreases at higher concentrations as, 0.1 µg/ml, 0.2 µg/ml and especially 1µg/ml, giving to bacteria a hard time for development.
+<center>https://static.igem.org/mediawiki/parts/9/96/UFAM_MERBA_8.png</center>
-The graph represented on Figure 2 shows the fluorescence emitted by DH5-alpha induced by different Hg concentrations in function of the time; and the graph represented on Figure 3, shows the ratio between fluorescence emitted and Optical Density.
-[[File:bs2.png]]
+After 18h, our construction reached 70% of mercury bioremediation! Want to see more? Access our wiki: 2016.igem.org/Team:UFAM-UEA_Brazil.
-'''Figure 2.''' GFP fluorescence intensity in the four given times, at mercury chloride concentrations of 0 µg/ml, 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml.
-[[File:bs3.png]]
-'''Figure 3.''' GFP fluorescence per cell growth ratio in the four given times, at mercury chloride concentrations of 0 µg/ml, 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml.
-Fluorescence can be observed in bacteria exposed to small concentrations, as 0.01µg/ml and 0.02µg/ml, but has low intensity. Fluorescence levels presents medium intensity in the higher concentration as consequence of cell death. Fluorescence intensity increased more than 480% in 0.2µg/ml concentrations, compared to fluorescence intensity from 1µg/ml, even in cell growth reduction, demonstrating its efficiency to induce mer promoter.
-'''Experiments and Results as a Biosensor (BBa_K1355001 + BBa_E0840)'''
-The experiment to quantify GFP expression induced by Hg was made according to the protocol “Quantification of Green Fluorescent Protein (GFP) induced by different concentrations of mercury in Escherichia coli DH5α”.
-DH5α transformed with BBa_K1355002 was inoculated in LM (LB with low concentration of NaCl) liquid medium with chloramphenicol and grew until the Optical Density was 0.4 to 0.6abs (measured on spectrophotometer at 600 nm wavelength). After cell growth, an aliquot of 500μl in 5 eppendorf tubes (2ml) was taken and then added mercury chloride in order to achieve the concentrations of: 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml. The samples were incubated at 37°C on shaker. We collected each eppendorf tube at time 1 (01:30 hours of incubation), time 2 (03:00 hours of incubation) and time 3 (04:30 hours of incubation). Every sample was centrifuged at 12000g for 3 minutes and the pellet washed with TN Buffer (Nacl 0.15M + Tris HCl 10mM) and then re-suspended with 500μl of the same buffer. The same process was made to the bacterium without construction as a control to GFP expression/intensity. GFP expression was measured using the Hidex Chameleon spectrofluorimeter with excitation filter 340 nm and emission filter 500 nm wavelength. The Optical Density was measured simultaneously. All samples were analyzed in triplicate.
-The graph represented on Figure 1 shows the Optical Density of transformed DH5α with BBa_K1355002 in different Hg concentrations in function of time:
-[[File:bs1.png]]
-'''Figure 1.''' Optical Density measured in the four given times, at mercury chloride concentrations of 0 µg/ml, 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml.
-In these condition cell growth increases along time. The highest values correspond to bacteria not exposed to mercury or to small concentrations, as in 0 µg/ml, 0.01 µg/ml and 0.02 µg/ml. Suggesting a harmless condition to bacteria. However, cell growth decreases at higher concentrations as, 0.1 µg/ml, 0.2 µg/ml and especially 1µg/ml, giving to bacteria a hard time for development.
-The graph represented on Figure 2 shows the fluorescence emitted by DH5-alpha induced by different Hg concentrations in function of the time; and the graph represented on Figure 3, shows the ratio between fluorescence emitted and Optical Density.
-[[File:bs2.png]]
-'''Figure 2.''' GFP fluorescence intensity in the four given times, at mercury chloride concentrations of 0 µg/ml, 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml.
-[[File:bs3.png]]
-'''Figure 3.''' GFP fluorescence per cell growth ratio in the four given times, at mercury chloride concentrations of 0 µg/ml, 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml.
-Fluorescence can be observed in bacteria exposed to small concentrations, as 0.01µg/ml and 0.02µg/ml, but has low intensity. Fluorescence levels presents medium intensity in the higher concentration as consequence of cell death. Fluorescence intensity increased more than 480% in 0.2µg/ml concentrations, compared to fluorescence intensity from 1µg/ml, even in cell growth reduction, demonstrating its efficiency to induce mer promoter.
 ===User Reviews===