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

UNIQe3026ad881787b28-partinfo-00000000-QINU UNIQe3026ad881787b28-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) = BBa_K1355002'''
+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 BIOACCUMULATOR (BBa_K1355001 + BBa_K346004) = BBa_K1355003'''
+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 for Hg bioaccumulation was made according to the protocol “Quantification of Mercury bio accumulated by metal binding peptide (MBP) in recombinant DH5-alpha in different Hg concentrations”. DH5-alpha transformed with BBa_K1355003 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 400μl was taken and distributed in 4 eppendorf tubes (1.5ml) and then added mercury chloride in order to achieve the concentrations: 50 ppb, 100 ppb, 250 ppb and 500 ppb. The samples were incubated at 37°C on shaker. We collected each eppendorf tube at time 1 (01:30 hours of incubation) and time 2 (03:00 hours of incubation). After the designated time, both were centrifuged at 12000g for 3 minutes and the supernatant recovered (LM medium). We washed the pellet with TN Buffer (Nacl 0.15M + Tris HCl 10mM) and then re-suspended with 400μl of the same buffer. To measure bio accumulated Hg, we need to quantify the Hg inside and outside of bacterium after the incubation/exposure time. So we collected and measured the amount of Hg in LM medium supernatant recovered and bacterium re-suspended in TN Buffer. For this we used the equipment Direct Mercury Analyzer (DMA-80). As a control to normal Hg bio accumulated in bacteria, we used DH5-alpha transformed with BBa_K1355002 (Hg bio detector device) which does not present the metal binding peptide. We also measured the Optical Density of each sample.
+<center>https://static.igem.org/mediawiki/parts/7/7e/UFAM_MERBA_10.png</center>
-The graph represented on Figure 1 shows the amount of Hg in supernatant (LM medium recovered) and in bacterium (DH5-alpha transformed with BBa_K1355003) at the time 1 (01:30 hours of incubation);
-[[File:bc1.png]]
+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!
-'''Figure 1:''' Metal binding peptide activity after 01:30 hours of incubation in five given concentrations of mercury chloride: 50 ppb, 100 ppb, 250 ppb e 500 ppb
+<center>https://static.igem.org/mediawiki/parts/e/ef/Ufam_uea_merr_part7.png</center>
-It can be observed that the amount of Hg in the Mercury Bacter bioaccumulator increases according to the raise of Hg concentration. The amount of Hg increased 22 times comparing the 50 ppb sample with 500 ppb sample. In the 500 ppb Hg concentration the control bacterium just accumulated 2 per cent of total Hg amount. On the contrary, Mercury Bacter accumulated 40 per cent of total Hg amount in just 01:30 hours of incubation!!!  The data keeps raising on the time 2! Check it out!
+<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).
-The graph represented on Figure 2 shows the amount of Hg in supernatant (LM medium recovered) and in bacterium (DH5-alpha transformed with BBa_K1355003) at the time 2 (03:00 hours of incubation);
+<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!
-[[File:bc2.png]]
+<center>https://static.igem.org/mediawiki/parts/9/96/UFAM_MERBA_8.png</center>
-'''Figure 2:''' Metal binding peptide activity after 03:00 hours of incubation in five given concentrations of mercury chloride: 50 ppb, 100 ppb, 250 ppb e 500 ppb.
+After 18h, our construction reached 70% of mercury bioremediation! Want to see more? Access our wiki: 2016.igem.org/Team:UFAM-UEA_Brazil.
-It can be observed that the amount of Hg in Mercury Bacter bioaccumulator, increases according to the time of incubation. The amount of Hg increased 30 times comparing the 50 ppb sample with 500 ppb sample. In the 500 ppb Hg concentration the control bacterium just accumulated 4% of total Hg amount. Instead, Mercury Bacter accumulated 58% of total Hg amount in just 03:00 hours of incubation!!!
-The data can be analyzed individually in each concentrations samples as shown in graphs represented in the figures below:
-[[File:bc3.png]]
-'''Figure 3:''' Metal binding peptide activity at 50 ppb in time 1 and 2;
-[[File:bc4.png]]
-'''Figure 4:''' Metal binding peptide activity at 100 ppb in time 1 and 2;
-[[File:bc5.png]]
-'''Figure 5:''' Metal binding peptide activity at 250 ppb in time 1 and 2;
-[[File:bc6.png]]
-'''Figure 6:''' Metal binding peptide activity at 500 ppb in time 1 and 2
-'''Experiments and Results as a BIOREMEDIATOR (BBa_K1355001 + BBa_K1355000) = BBa_K1355004'''
-The experiment for Hg bioremediation was made according to the protocol “Cell growth quantification of mercury resistant Escherichia coli DH5α at differents Hg concentrations by Optical Density”.
-DH5-alpha transformed with BBa_K1355004 was inoculated in LM (LB with low concentration of NaCl) liquid medium with 0.05μg/ml of mercury chloride and 34μg/ml of chloramphenicol. The inoculum was incubated in shaker overnight at 37°C. After cell growth, we measured Optical Density (O.D.) of the cell culture in spectrophotometer at 600 nm wavelength. A standard quantity aliquot of bacterial suspension was taken in six falcons (50ml) with 10 ml of LM liquid medium and then added mercury chloride in order to achieve the concentrations: 0μg/ml, 1 μg/ml; 2.5 μg/ml; 5 μg/ml; 10μg/ml; 20 μg/ml. The samples were incubated at 37°C on shaker. We collected each sample at the given times, 1 (02:30 hours of incubation), time 2 (04:30 hours of incubation), time 3 (06:30 hours of incubation), time 4 (17 hours of incubation), time 5 (24 hours of incubation) to measure O.D. on spectrophotometer. As control, we used DH5-alpha transformed with BBa_K1355002 (Hg bio detector device) which does not present coding regions for the mercuric ion reductase. We also measured Optical Density of each sample. After cell growth measurement, the samples were frozen to subsequently quantification of Hg remediated by the Mercury Bacter.
-The graph represented on Figure 1 and Figure 2 shows Cell growth of Mercury Bacter and of the control bacteria in different Hg concentrations in function of time, respectively;
-[[File:br1.png]]
-'''Figure 1:''' Cell growth of Mercury Bacter in different Hg concentrations in function of time;
-[[File:br2.png]]
-'''Figure 2:''' Cell growth of control bacteria in different Hg concentrations in function of time
-Comparing the graphs shown on figure 1 and 2, it can be observed that the biobrick BBa_K135504 provided resistance to high Hg concentrations. Control bacteria did not present resistance to Hg concentration equal or higher to 1 ppm. On the contrary, Mercury Bacter grew up even in 10 ppm Hg concentrations. Interestingly, at the concentration of 10 ppm Mercury Bacter needed 24 hours to exhibit increase in cell growth, and after 48 hours it maintained the same OD, unlike the others. At 1 ppm, 2.5 ppm and 5 ppm Mercury Bacter performed an overwhelming cellular growth after 24 hours of exposure to Hg, reaching approximately the same cellular growth as the non-exposed bacteria!!
-The graph represented on Figure 3 compares Hg concentration in LM medium after 48 hours of experiment, between medium with bacteria used as control and engineered Mercury Bacter.
-[[File:br3.png]]
-'''Figure 3:''' Hg concentration in LM medium after 48 hours of cell growth in control bacteria and in the genetically engineered Mercury Bacter.
-In the graph above it can be observed that our super Mercury Bacter bioremediated! In action, Mercury Bacter reduced 72% of the mercury levels, in comparison to control at the sample 5 ppm! In 1 ppm,  Even though on 10 ppm concentration, where bacteria had a slow growth rate only after 24 hours, it bioremediated 48.02%! These results demonstrate  our constructions’ efficiency, both for biobrick BBa_K1355001 as a promoter regulated by MerR protein and to BBa_K1355000 as MerA encoding gene!
 ===User Reviews===