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PROJECT.html
From 2012.igem.org
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.
