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<div id="text1" style="position:absolute; overflow:hidden; left:198px; top:92px; width:701px; height:87px; z-index:2"> | <div id="text1" style="position:absolute; overflow:hidden; left:198px; top:92px; width:701px; height:87px; z-index:2"> | ||
<div class="wpmd"> | <div class="wpmd"> | ||
| - | <div align=center style="line-height:1.50;"><font color="#99CC00" face="Calibri" class=" | + | <div align=center style="line-height:1.50;"><font color="#99CC00" face="Calibri" class="ws22">Genetically Modified </font><font color="#99CC00" face="Calibri" class="ws22"><I>E. coli</I></font><font color="#99CC00" face="Calibri" class="ws22"> as an Alternative Biosensor of Cyanide and Cyanide Compounds</font></div> |
| - | <div align=center style="line-height:1.50;"><font color="#99CC00" face="Calibri" class=" | + | <div align=center style="line-height:1.50;"><font color="#99CC00" face="Calibri" class="ws22"><BR></font></div> |
<div align=center><font face="Calibri" class="ws24"><BR></font></div> | <div align=center><font face="Calibri" class="ws24"><BR></font></div> | ||
</div></div> | </div></div> | ||
| - | <div id="text2" style="position:absolute; overflow:hidden; left:133px; top:192px; width:815px; height: | + | <div id="text2" style="position:absolute; overflow:hidden; left:133px; top:192px; width:815px; height:472px; z-index:3"> |
<div class="wpmd"> | <div class="wpmd"> | ||
| - | <div><font color="#808080" face="Calibri" class="ws14">Cyanide is considered an extremely harmful toxic for the environment and living organisms since it inhibits the cellular respiration at the level of electron transport chain. In the industrial sector, cyanide is used to produce paper, paints, textiles and plastics. It is also very common in the mining industry as a way to recover metals. Due to its application and toxicity, it is necessary to monitor and keep the cyanide at a subtoxic level. </font></div> | + | <div align=center><font color="#808080" face="Calibri" class="ws14"><B>ABSTRACT</B></font></div> |
| - | <div><font color="#808080" face="Calibri" class="ws14"><BR></font></div> | + | <div align=center><font color="#808080" face="Calibri" class="ws14"><B><BR></B></font></div> |
| - | <div><font color="#808080" face="Calibri" class="ws14">We will incorporate genes that will allow the bacteria to become a biosensor with the capacity to detect the presence of cyanide and cyanide compounds by adding the expression of a reporter gene (RFP) under the control of a promoter inducible by these compounds. This gene comes from the bacteria </font><font color="#808080" face="Calibri" class="ws14"><I>Pseudomonas pseudoalcaligenes</I></font><font color="#808080" face="Calibri" class="ws14">. This new tecnique, which will be used to detect water and soil contamination, will also become a platform so that in the future we could incorporate a gene that allows the bacteria, not only detect, but also degrade these compounds using a method that is accessible and environmentally friendly through bioremediation. </font></div> | + | <div align=justify><font color="#808080" face="Calibri" class="ws14">Cyanide is considered an extremely harmful toxic for the environment and living organisms since it inhibits the cellular respiration at the level of electron transport chain. In the industrial sector, cyanide is used to produce paper, paints, textiles and plastics. It is also very common in the mining industry as a way to recover metals. Due to its application and toxicity, it is necessary to monitor and keep the cyanide at a subtoxic level. </font></div> |
| + | <div align=justify><font color="#808080" face="Calibri" class="ws14"><BR></font></div> | ||
| + | <div align=justify><font color="#808080" face="Calibri" class="ws14">We will incorporate genes that will allow the bacteria to become a biosensor with the capacity to detect the presence of cyanide and cyanide compounds by adding the expression of a reporter gene (RFP) under the control of a promoter inducible by these compounds. This gene comes from the bacteria </font><font color="#808080" face="Calibri" class="ws14"><I>Pseudomonas pseudoalcaligenes</I></font><font color="#808080" face="Calibri" class="ws14">. This new tecnique, which will be used to detect water and soil contamination, will also become a platform so that in the future we could incorporate a gene that allows the bacteria, not only detect, but also degrade these compounds using a method that is accessible and environmentally friendly through bioremediation. </font></div> | ||
| + | <div align=justify><font color="#808080" face="Calibri" class="ws14"><BR></font></div> | ||
| + | <div align=justify><font color="#808080" face="Calibri" class="ws14">In order for the bacteria to degrade cyanide, it needs not only the metabolic route, but a sort of resistance to these compounds. For this reason, we will also add cyanide resistant genes (cioAB) to elevate the detection potential of our biosensor. This will provide the bacteria an alternate route for the electron transportation insensitive to cyanide. </font></div> | ||
| + | </div></div> | ||
| + | |||
| + | <div id="text3" style="position:absolute; overflow:hidden; left:139px; top:698px; width:815px; height:344px; z-index:5"> | ||
| + | <div class="wpmd"> | ||
| + | <div align=center><font color="#808080" face="Calibri" class="ws14"><B>OBJECTIVES</B></font></div> | ||
| + | <UL> | ||
| + | <li style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14">Build a resistance expression platform for cyanide compounds.</font></li> | ||
| + | <li style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14">Build a detection platform for cyanide compounds. </font></li> | ||
| + | <li style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14">Finish our functional cyanide biosensor with resistance and detection genes. </font></li> | ||
| + | </UL> | ||
| + | <div align=justify><font color="#808080" face="Calibri" class="ws14"><BR></font></div> | ||
| + | <div align=center><font color="#808080" face="Calibri" class="ws14"><B>SPECIFIC OBJECTIVES</B></font></div> | ||
| + | <div align=center><font color="#808080" face="Calibri" class="ws14"><B><BR></B></font></div> | ||
| + | <UL> | ||
| + | <li><font color="#808080" face="Calibri" class="ws14">Assemble a constitutive promoter with an RBS and with CioAB genes, together with GFP + Ter for the resistance expression platform. </font></li> | ||
| + | <li><font color="#808080" face="Calibri" class="ws14">Assemble an inducible promoter with an RBS, together with RFP + Ter for the detection expression platform. </font></li> | ||
| + | <li><font color="#808080" face="Calibri" class="ws14">Build a functional biosensor assembling these two parts and characterize its performance. </font></li> | ||
| + | </UL> | ||
| + | <div align=justify><font color="#808080" face="Calibri" class="ws14"><BR></font></div> | ||
<div><font color="#808080" face="Calibri" class="ws14"><BR></font></div> | <div><font color="#808080" face="Calibri" class="ws14"><BR></font></div> | ||
| - | |||
</div></div> | </div></div> | ||
| - | <div id=" | + | <div id="image2" style="position:absolute; overflow:hidden; left:496px; top:1323px; width:133px; height:196px; z-index:6"><img src="https://static.igem.org/mediawiki/2012/8/82/Paste30.jpg" alt="" title="" border=0 width=133 height=196></div> |
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| - | </div> | + | <div id="text4" style="position:absolute; overflow:hidden; left:359px; top:1531px; width:410px; height:82px; z-index:7"> |
| + | <div class="wpmd"> | ||
| + | <div align=center><font color="#808080" face="Calibri" class="ws14"><B><BR></B></font></div> | ||
| + | <div><font color="#808080" face="Calibri" class="ws14"> </font><font color="#808080" face="Calibri" class="ws12"><I>** The ingestion of 50 to 100mg of sodium cyanide or potassium cyanide is immediately follow by unconsciousness and respiratory arrest!!**</I></font></div> | ||
| + | </div></div> | ||
| + | |||
| + | <div id="text5" style="position:absolute; overflow:hidden; left:159px; top:1645px; width:816px; height:469px; z-index:8"> | ||
| + | <div class="wpmd"> | ||
| + | <div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14">We are generating a new alternative for detecting cyanide and cyanide compound in water and soil with potentially contaminated from mining or other industrial activities. Here in Panama the mining industry represents 1.8% of our Gross Domestic Product (GDP), totaling around 344.1 million dollars (2011). In 1998 there was a major cyanide spill that caused the dead of thousands of fishes and endangered the lives of many people. </font></div> | ||
| + | <div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14">Due to its spread use and toxicity, as we just mentioned, we think that it is necessary to monitor and keep the cyanide at a subtoxic level.</font></div> | ||
| + | <div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14">In this project, we are generating a new alternative to measure the levels of this toxic product by using genetic engineering to modify E. coli at the level of DNA. We will incorporate genes that will allow the bacteria to become a biosensor itself, with the capacity to detect the presence of cyanide and cyanide compounds by adding the expression of a reporter gene (RFP) under the control of a promotor inducible by these compounds. This gene comes from an inducible enzyme from the bacteria </font><font color="#808080" face="Calibri" class="ws14"><I>Pseudomonas pseudoalcaligenes.</I></font><font color="#808080" face="Calibri" class="ws14"> In order for the bacteria to degrade cyanide, it needs not only the metabolic route, but a sourt of resistance to the lethal action of these compounds. For this reason, we will also add cyanide resistant genes (cioAB) to elevate the detection potential of our biosensor. They will provide the bacteria with an alternate route for the electron transport, insensitive to cyanide. </font></div> | ||
| + | </div></div> | ||
| + | |||
| + | <div id="text6" style="position:absolute; overflow:hidden; left:162px; top:2620px; width:815px; height:296px; z-index:9"> | ||
| + | <div class="wpmd"> | ||
| + | <div align=center style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><B>FUTURE WORK</B></font></div> | ||
| + | <div align=center style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><B><BR></B></font></div> | ||
| + | <div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14">This new technique, which will be used to detect water and soil contamination, will also become a platform for our future work, where we plan to incorporate other genes such as the CynS gene that encodes for an enzyme called cyanase. This enzyme is used for different microorganisms to degrade cyanate into ammonium and CO2 so we would have a cyanide biodegrading tool. There is lot of ongoing research trying to fiound the exact pathway for cyanide degradation in bacteria; if this is found and characterized, we could also incorporate these genes into our biobrick, this will allowing our bacteria, not only to detect, but also to degrade these compounds using a method that is accessible and environmentally friendly acting in bioremediation.</font></div> | ||
| + | </div></div> | ||
| + | |||
| + | <div id="image3" style="position:absolute; overflow:hidden; left:337px; top:2118px; width:409px; height:233px; z-index:10"><img src="https://static.igem.org/mediawiki/2012/b/ba/Paste31.jpg" alt="" title="" border=0 width=409 height=233></div> | ||
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| + | <div id="image4" style="position:absolute; overflow:hidden; left:517px; top:2358px; width:47px; height:125px; z-index:11"><img src="https://static.igem.org/mediawiki/2012/d/da/Paste32.jpg" alt="" title="" border=0 width=47 height=125></div> | ||
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| + | <div id="image5" style="position:absolute; overflow:hidden; left:427px; top:2496px; width:200px; height:102px; z-index:12"><img src="https://static.igem.org/mediawiki/2012/3/35/Paste33.jpg" alt="" title="" border=0 width=200 height=102></div> | ||
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| + | <div id="text7" style="position:absolute; overflow:hidden; left:159px; top:3016px; width:815px; height:443px; z-index:13"> | ||
| + | <div class="wpmd"> | ||
| + | <div align=center style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><B>REFERENCES</B></font></div> | ||
| + | <div align=center style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><B><BR></B></font></div> | ||
| + | <OL> | ||
| + | <li value=1 style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14">Pictures and info taken from: Supramolecular organization of protein complexes in the mitochondrial inner membrane. Janet Vonck, Eva Schäfer. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research</font></li> | ||
| + | <li value=2 style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14">Volume 1793, Issue 1, January 2009, Pages 117-124</font></li> | ||
| + | <li value=3 style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14">Assembly of the Mitochondrial Respiratory Chain http://www.sciencedirect.com/science/article/pii/S0167488908002012</font></li> | ||
| + | <li value=4 style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14">http://www.marisolcollazos.es/noticias-criminologia/?p=6547</font></li> | ||
| + | <li value=5 style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14">http://www-cs-faculty.stanford.edu/~eroberts/courses/ww2/projects/chemical-biological-warfare/cyanide.htm</font></li> | ||
| + | <li value=6 style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14">The Role of Cyanide in Smoke Inhalation: New Treatment for a Silent Killer (Slides with Transcript)</font></li> | ||
| + | <li value=7 style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14">Gregory M Bogdan, PhD; Donald W Walsh, PhD, EMT-P; Marc Eckstein, MD, MPH, FACEP http://www.medscape.org/viewarticle/559849_2</font></li> | ||
| + | </OL> | ||
| + | <div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><BR></font></div> | ||
| + | <div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14"><BR></font></div> | ||
| + | </div></div> | ||
| + | |||
| + | <div id="text8" style="position:absolute; overflow:hidden; left:127px; top:1024px; width:878px; height:287px; z-index:14"> | ||
| + | <div class="wpmd"> | ||
| + | <div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14">Cyanide is considered an extremely harmful toxic to both the environment and living organisms. In the industrial sector, cyanide is used to produce paper, paints, textiles and plastics. It is also very common in the mining industry used for metal extraction and recovery. Aqueous solutions of cyanide are able to complex with gold and silver to form compounds that remain soluble in alkaline solutions of cyanide salts. Once the desired metal has moved into the solution by complexing to the cyanide, the metal is extracted. The most commonly used cyanide salts in the mining industry are sodium cyanide (NaCN), potassium cyanide (KCN) and calcium cyanide (Ca(CN)2). </font></div> | ||
| + | <div align=justify style="line-height:1.50;"><font color="#808080" face="Calibri" class="ws14">The percentage of cyanide converted to cyanic acid (HCN) in aqueous solutions of cyanide is dependent on the pH of the solution;, it is necessary for the pHs levels to be more than 10.5 to prevent the formation and release of the gas form of HCN which is the one that we inhale. </font></div> | ||
| + | </div></div> | ||
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Latest revision as of 23:04, 26 September 2012
Genetically Modified E. coli as an Alternative Biosensor of Cyanide and Cyanide Compounds
ABSTRACT
Cyanide is considered an extremely harmful toxic for the environment and living organisms since it inhibits the cellular respiration at the level of electron transport chain. In the industrial sector, cyanide is used to produce paper, paints, textiles and plastics. It is also very common in the mining industry as a way to recover metals. Due to its application and toxicity, it is necessary to monitor and keep the cyanide at a subtoxic level.
We will incorporate genes that will allow the bacteria to become a biosensor with the capacity to detect the presence of cyanide and cyanide compounds by adding the expression of a reporter gene (RFP) under the control of a promoter inducible by these compounds. This gene comes from the bacteria Pseudomonas pseudoalcaligenes. This new tecnique, which will be used to detect water and soil contamination, will also become a platform so that in the future we could incorporate a gene that allows the bacteria, not only detect, but also degrade these compounds using a method that is accessible and environmentally friendly through bioremediation.
In order for the bacteria to degrade cyanide, it needs not only the metabolic route, but a sort of resistance to these compounds. For this reason, we will also add cyanide resistant genes (cioAB) to elevate the detection potential of our biosensor. This will provide the bacteria an alternate route for the electron transportation insensitive to cyanide.
OBJECTIVES
- Build a resistance expression platform for cyanide compounds.
- Build a detection platform for cyanide compounds.
- Finish our functional cyanide biosensor with resistance and detection genes.
SPECIFIC OBJECTIVES
- Assemble a constitutive promoter with an RBS and with CioAB genes, together with GFP + Ter for the resistance expression platform.
- Assemble an inducible promoter with an RBS, together with RFP + Ter for the detection expression platform.
- Build a functional biosensor assembling these two parts and characterize its performance.
** The ingestion of 50 to 100mg of sodium cyanide or potassium cyanide is immediately follow by unconsciousness and respiratory arrest!!**
We are generating a new alternative for detecting cyanide and cyanide compound in water and soil with potentially contaminated from mining or other industrial activities. Here in Panama the mining industry represents 1.8% of our Gross Domestic Product (GDP), totaling around 344.1 million dollars (2011). In 1998 there was a major cyanide spill that caused the dead of thousands of fishes and endangered the lives of many people.
Due to its spread use and toxicity, as we just mentioned, we think that it is necessary to monitor and keep the cyanide at a subtoxic level.
In this project, we are generating a new alternative to measure the levels of this toxic product by using genetic engineering to modify E. coli at the level of DNA. We will incorporate genes that will allow the bacteria to become a biosensor itself, with the capacity to detect the presence of cyanide and cyanide compounds by adding the expression of a reporter gene (RFP) under the control of a promotor inducible by these compounds. This gene comes from an inducible enzyme from the bacteria Pseudomonas pseudoalcaligenes. In order for the bacteria to degrade cyanide, it needs not only the metabolic route, but a sourt of resistance to the lethal action of these compounds. For this reason, we will also add cyanide resistant genes (cioAB) to elevate the detection potential of our biosensor. They will provide the bacteria with an alternate route for the electron transport, insensitive to cyanide.
FUTURE WORK
This new technique, which will be used to detect water and soil contamination, will also become a platform for our future work, where we plan to incorporate other genes such as the CynS gene that encodes for an enzyme called cyanase. This enzyme is used for different microorganisms to degrade cyanate into ammonium and CO2 so we would have a cyanide biodegrading tool. There is lot of ongoing research trying to fiound the exact pathway for cyanide degradation in bacteria; if this is found and characterized, we could also incorporate these genes into our biobrick, this will allowing our bacteria, not only to detect, but also to degrade these compounds using a method that is accessible and environmentally friendly acting in bioremediation.
REFERENCES
- Pictures and info taken from: Supramolecular organization of protein complexes in the mitochondrial inner membrane. Janet Vonck, Eva Schäfer. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
- Volume 1793, Issue 1, January 2009, Pages 117-124
- Assembly of the Mitochondrial Respiratory Chain http://www.sciencedirect.com/science/article/pii/S0167488908002012
- http://www.marisolcollazos.es/noticias-criminologia/?p=6547
- http://www-cs-faculty.stanford.edu/~eroberts/courses/ww2/projects/chemical-biological-warfare/cyanide.htm
- The Role of Cyanide in Smoke Inhalation: New Treatment for a Silent Killer (Slides with Transcript)
- Gregory M Bogdan, PhD; Donald W Walsh, PhD, EMT-P; Marc Eckstein, MD, MPH, FACEP http://www.medscape.org/viewarticle/559849_2
Cyanide is considered an extremely harmful toxic to both the environment and living organisms. In the industrial sector, cyanide is used to produce paper, paints, textiles and plastics. It is also very common in the mining industry used for metal extraction and recovery. Aqueous solutions of cyanide are able to complex with gold and silver to form compounds that remain soluble in alkaline solutions of cyanide salts. Once the desired metal has moved into the solution by complexing to the cyanide, the metal is extracted. The most commonly used cyanide salts in the mining industry are sodium cyanide (NaCN), potassium cyanide (KCN) and calcium cyanide (Ca(CN)2).
The percentage of cyanide converted to cyanic acid (HCN) in aqueous solutions of cyanide is dependent on the pH of the solution;, it is necessary for the pHs levels to be more than 10.5 to prevent the formation and release of the gas form of HCN which is the one that we inhale.
