Team:Cornell/project/wetlab/mercury

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<h1 style="padding: 0px; margin-bottom: 0px;">Wetlab</h1>
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<p>Mercury</p>
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<li><a href="https://2014.igem.org/Team:Cornell/project/wetlab">Overview</a></li>
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<li class="active"><a href="https://2014.igem.org/Team:Cornell/project/wetlab/mercury">Mercury</a></li>
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<li><a href="https://2014.igem.org/Team:Cornell/project/wetlab/nickel">Nickel</a></li>
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<li><a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein">Metallothioneins</a></li>
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<h1 style="margin-top: 0px;">Header 1</h1>
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<h1>Construct Design</h1>
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Text text text Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt.  
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To allow for the transport and sequestration of mercury ions into <i>E. coli</i> cells, genes that encode for the cellular production of heavy metal transport proteins and metallothioneins have been added to the pSB1C3 high copy bacterial plasmid. The mercury transport system is composed of <i>merT</i> and <i>merP</i>, genes originally found in <i>Pseudomonas aeruginosa</i>. <i>merP</i> is a periplasmic mercury ion scavenging protein. <i>merT</i> is an integrated membrane protein that works to transport mercury ions into the cell’s cytoplasm.<sup>[1]</sup> The <i>merT</i> and <i>merP</i> coupled transport system has been used in previous studies to develop luminescence based biosensors for the detection of mercury in the surroundings of bacterial cells.<sup>[2]</sup>
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Our BioBrick BBa_K1460004 is composed of the Anderson promoter followed by a ribosomal binding site, <i>merT</i>, <i>merP</i>, and a terminator. The constitutive  Anderson promoter allows for the constant expression of metal uptake proteins within our engineered <i>E. coli</i>. The BioBrick <a href="http://parts.igem.org/Part:BBa_K1460007">BBa_K1460007</a> is a composite of parts <a href="http://parts.igem.org/Part:BBa_K1460004">BBa_K1460004</a> and <a href="http://parts.igem.org/Part:BBa_K1460001">BBa_K1460001</a>, and it contains the mercury transport proteins (along with promoter, ribosomal binding site, and terminator) upstream of the <i>GST-crs5</i> metallothionein gene in pSB1C3. By coupling the <i>merT</i> and <i>merP</i> system with metallothionein, we hope to develop an effective biological system for our cells to uptake mercury ions and bind intracellularly to metallothioneins.
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Far far away, behind the word mountains, far from the countries Vokalia and Consonantia, there live the blind texts. Separated they live in Bookmarksgrove right at the coast of the Semantics, a large language ocean. A small river named Duden flows by their place and supplies it with the necessary regelialia.
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<h3>BBa_K1460007</h3>
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Far far away, behind the word mountains, far from the countries Vokalia and Consonantia, there live the blind texts. Separated they live in Bookmarksgrove right at the coast of the Semantics, a large language ocean. A small river named Duden flows by their place and supplies it with the necessary regelialia.
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But I must explain to you how all this mistaken idea of denouncing pleasure and praising pain was born and I will give you a complete account of the system, and expound the actual teachings of the great explorer of the truth, the master-builder of human happiness.
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<h1>Results</h1>
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Cells successfully expressing <i>merT</i> and <i>merP</i> should be transporting more mercury ions past the cell wall. This would lead to increased mercury sensitivity. Additionally, cells expressing <i>merT</i> and <i>merP</i> as well as metallothionein should have increased tolerance to mercury due to the presence of metallothionein.  To test for mercury sensitivity, <i>E.coli</i> BL21 and engineered BL21  with part BBa_K1460004 in the cm<sup>r</sup> plasmid pSB1C3 were grown for a 24 hour period in LB with .5 uM Hg (a mercury concentration we found to have moderate toxicity to wild type BL21 cells).  To test for increased metal tolerance, we grew <i>E.coli</i> BL21  and engineered BL21  with parts BBa_K1460001 (<i>GST-crs5</i> in pSB1C3) and BBa_K1460004 (<i>merT/merP</i> in pUC57) in 5 uM Hg (a mercury concentration we found to be very toxic to wild type BL21 cells).
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The quick, brown fox jumps over a lazy dog. DJs flock by when MTV ax quiz prog. Junk MTV quiz graced by fox whelps. Bawds jog, flick quartz, vex nymphs. Waltz, bad nymph, for quick jigs vex! Fox nymphs grab quick-jived waltz. Brick quiz whangs jumpy veldt fox. Bright vixens jump; dozy fowl quack.
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What we observe in both cases is what we expect. We see that BL21 engineered with BBa_K1460004 has impaired growth when compared to wild type BL21 (figure 1). This suggests that, in fact, BBa_K1460004 acts as expected and engineered cells successfully transport more mercury ions past the membrane than wild type cells. When BL21 engineered with both <i>merT/merP</i> and <i>GST-crs5</i> are grown in a highly toxic concentration of mercury we see significant growth when in wild type BL21 we do not (figure 2). This suggests that these cells are successfully expressing metallothionein and that this metallothionein is providing the cells with an inherent resistance to mercury toxicity.
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Part BBa_K1460004 in pUC57 was co-transformed with part BBa_K1460001 (<i>GST-crs5</i>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the mercury sequestration part BBa_K1460007. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460004 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Hg for a final mercury concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for mercury concentration using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) with the help of Cornell's Nutrient Analysis Lab. Error bars in chart represent standard deviation of three biological replicates.
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No one rejects, dislikes, or avoids pleasure itself, because it is pleasure, but because those who do not know how to pursue pleasure rationally encounter consequences that are extremely painful. Nor again is there anyone who loves or pursues or desires to obtain pain of itself, because it is pain, but because occasionally circumstances occur in which toil and pain can procure him some great pleasure. To take a trivial example, which of us ever undertakes laborious physical exercise, except to obtain some advantage from it? But who has any right to find fault with a man who chooses to enjoy a pleasure that has no annoying consequences, or one who avoids a pain that produces no resultant pleasure? On the other hand, we denounce with righteous indignation and dislike men who are so beguiled and demoralized by the charms of pleasure of the moment, so blinded by desire, that they cannot foresee.
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There was no statistically significant difference between BL21 wild type and BL21 engineered to express <i>merT/merP</i> and <i>GST-crs5</i> in final culture concentration of mercury or mercury sequestered per OD. This result prevents us from definitively confirming that the engineered bacteria are capable of sequestering mercury. The mercury concentrations used in this test were much higher than was shown in growth experiments to completely prevent growth of BL21, so it is likely that cells were quickly killed once metal was added, possibly confounding results. To verify this construct is successful in removing mercury from water, we must repeat these experiments using lower concentrations of Hg.  We were not able to complete these experiments, however, as the limit of detection of the ICP-AES used to test these metal concentrations is above the uM range necessary to conduct these experiments.
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The quick, brown fox jumps over a lazy dog. DJs flock by when MTV ax quiz prog. Junk MTV quiz graced by fox whelps. Bawds jog, flick quartz, ex nymphs. Waltz, bad nymph, for quick jigs vex! Fox nymphs grab quick-jived waltz. Brick quiz whangs jumpy veldt fox. Bright vixens jump; dozy fowl quack.
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<li>Ref 1</li>
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<li>Lund, P., & Brown, N. (1987). Role of the <i>merT</i> and <i>merP</i> gene products of transposon Tn501 in the induction and expression of resistance to mercuric ions. <i>Gene</i>, 207-214.</li>
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<li>Ref 2</li>
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<li>Omura, T., Kiyono, M., & Pan-Hou, H. (2004). Development of a Specific and Sensitive Bacteria Sensor for Detection of Mercury at Picomolar Levels in Environment. <i>Journal of Health Science</i>, 379-383.</li>
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Latest revision as of 03:52, 18 October 2014

Cornell iGEM

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Wet Lab

Construct Design

To allow for the transport and sequestration of mercury ions into E. coli cells, genes that encode for the cellular production of heavy metal transport proteins and metallothioneins have been added to the pSB1C3 high copy bacterial plasmid. The mercury transport system is composed of merT and merP, genes originally found in Pseudomonas aeruginosa. merP is a periplasmic mercury ion scavenging protein. merT is an integrated membrane protein that works to transport mercury ions into the cell’s cytoplasm.[1] The merT and merP coupled transport system has been used in previous studies to develop luminescence based biosensors for the detection of mercury in the surroundings of bacterial cells.[2]

Our BioBrick BBa_K1460004 is composed of the Anderson promoter followed by a ribosomal binding site, merT, merP, and a terminator. The constitutive Anderson promoter allows for the constant expression of metal uptake proteins within our engineered E. coli. The BioBrick BBa_K1460007 is a composite of parts BBa_K1460004 and BBa_K1460001, and it contains the mercury transport proteins (along with promoter, ribosomal binding site, and terminator) upstream of the GST-crs5 metallothionein gene in pSB1C3. By coupling the merT and merP system with metallothionein, we hope to develop an effective biological system for our cells to uptake mercury ions and bind intracellularly to metallothioneins.

BBa_K1460007

Results

Cells successfully expressing merT and merP should be transporting more mercury ions past the cell wall. This would lead to increased mercury sensitivity. Additionally, cells expressing merT and merP as well as metallothionein should have increased tolerance to mercury due to the presence of metallothionein. To test for mercury sensitivity, E.coli BL21 and engineered BL21 with part BBa_K1460004 in the cmr plasmid pSB1C3 were grown for a 24 hour period in LB with .5 uM Hg (a mercury concentration we found to have moderate toxicity to wild type BL21 cells). To test for increased metal tolerance, we grew E.coli BL21 and engineered BL21 with parts BBa_K1460001 (GST-crs5 in pSB1C3) and BBa_K1460004 (merT/merP in pUC57) in 5 uM Hg (a mercury concentration we found to be very toxic to wild type BL21 cells).
What we observe in both cases is what we expect. We see that BL21 engineered with BBa_K1460004 has impaired growth when compared to wild type BL21 (figure 1). This suggests that, in fact, BBa_K1460004 acts as expected and engineered cells successfully transport more mercury ions past the membrane than wild type cells. When BL21 engineered with both merT/merP and GST-crs5 are grown in a highly toxic concentration of mercury we see significant growth when in wild type BL21 we do not (figure 2). This suggests that these cells are successfully expressing metallothionein and that this metallothionein is providing the cells with an inherent resistance to mercury toxicity.



Part BBa_K1460004 in pUC57 was co-transformed with part BBa_K1460001 (GST-crs5) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the mercury sequestration part BBa_K1460007. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460004 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Hg for a final mercury concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for mercury concentration using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) with the help of Cornell's Nutrient Analysis Lab. Error bars in chart represent standard deviation of three biological replicates.
There was no statistically significant difference between BL21 wild type and BL21 engineered to express merT/merP and GST-crs5 in final culture concentration of mercury or mercury sequestered per OD. This result prevents us from definitively confirming that the engineered bacteria are capable of sequestering mercury. The mercury concentrations used in this test were much higher than was shown in growth experiments to completely prevent growth of BL21, so it is likely that cells were quickly killed once metal was added, possibly confounding results. To verify this construct is successful in removing mercury from water, we must repeat these experiments using lower concentrations of Hg. We were not able to complete these experiments, however, as the limit of detection of the ICP-AES used to test these metal concentrations is above the uM range necessary to conduct these experiments.

References


  1. Lund, P., & Brown, N. (1987). Role of the merT and merP gene products of transposon Tn501 in the induction and expression of resistance to mercuric ions. Gene, 207-214.
  2. Omura, T., Kiyono, M., & Pan-Hou, H. (2004). Development of a Specific and Sensitive Bacteria Sensor for Detection of Mercury at Picomolar Levels in Environment. Journal of Health Science, 379-383.