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Team:Queens Canada/Project/Repel
From 2013.igem.org
QGEM 2013 PRESENTS:
Mosquito Repellent
Our idea for a mosquito repellent is to neutralize the odours that mosquitoes are attracted to. Recent studies have shown that malarial mosquitoes rely 4x more heavily on their sense of smell to find their prey. By removing the volatile compounds that cause these smells, we are effectively able to decrease the ability of mosquitoes to find humans. To achieve this end, we have created a genetic and enzymatic pathway. It begins with the uptake of isovaleric acid, the smell inducing compound, and converts it into a pleasant banana odour. Domestically, this serves as a foot deodorant but on a global scale it may also curb malaria rates.
AtoE
AtoE is a non-specific membrane transporter of short-chain fatty acids endogenously expressed in E. coli. High constitutive expression of this gene would allow high intake of surrounding isovaleric acid and channelling into the breakdown pathway.
In order to isolate the gene from the E. coli genome, we designed PCR primers such that the AtoE gene could be replicated with the biobrick cut sites on the ends of it. The size of the AtoE gene is 1323bp. With the addition of 43bp from the standard biobrick prefix and suffix, we should get a band around 1.4kb as shown by the gel below of the PCR products we got.
In order to isolate the gene from the E. coli genome, we designed PCR primers such that the AtoE gene could be replicated with the biobrick cut sites on the ends of it. The size of the AtoE gene is 1323bp. With the addition of 43bp from the standard biobrick prefix and suffix, we should get a band around 1.4kb as shown by the gel below of the PCR products we got.
AtoE is a non-specific membrane transporter of short-chain fatty acids endogenously expressed in E. coli. High constitutive expression of this gene would allow high intake of surrounding isovaleric acid and channelling into the breakdown pathway.
In order to isolate the gene from the E. coli genome, we designed PCR primers such that the AtoE gene could be replicated with the biobrick cut sites on the ends of it. The size of the AtoE gene is 1323bp. With the addition of 43bp from the standard biobrick prefix and suffix, we should get a band around 1.4kb as shown by the gel below of the PCR products we got.
In order to isolate the gene from the E. coli genome, we designed PCR primers such that the AtoE gene could be replicated with the biobrick cut sites on the ends of it. The size of the AtoE gene is 1323bp. With the addition of 43bp from the standard biobrick prefix and suffix, we should get a band around 1.4kb as shown by the gel below of the PCR products we got.
NCAR and NPT
Carboxylic acid reductase (CAR) is an enzyme that converts fatty acids such as isovaleric into aldehydes. Phosphopantetheinyl transferase (NPT) is an enzyme that induces a post-translational modification by adding a pantetheinyl arm to the active site of the carboxylic acid reductase, which is crucial for the formation of a thioester intermediate.
YjgB
YjgB is non-specific zinc-containing alcohol dehydrogenase endogenously expressed in E. coli. It will reduce isoamyl aldehyde into isoamyl alcohol.
YjgB is the part that is being submitted this year. More details on this can be found on the Parts page on the wiki.
YjgB is the part that is being submitted this year. More details on this can be found on the Parts page on the wiki.
ATF1
Alcohol acetyl-transferase 1 (ATF1) (BBa_J45014) is a enzyme endogenous to Saccharomyces cerevisiae which converts isoamyl alcohol to isoamyl acetate. This will complete the neutralization of isovaleric acid. This enzyme was first used by MIT iGEM 2006. We received the ATF gene from the biobrick registry where we transformed and cut the part to be placed in with the other genes in the pathway.
Testing Isovaleric Acid
Caenorhabditis elegans has impressive chemotaxis behaviour, used naturally to seek out food sources. It has been shown to respond to a wide range of both water-soluble and volatile chemicals with a GPCR system, as either attractants or repellants.
In order to test the effectiveness of our part, it was necessary to create an assay to test our completed part against. Using the assay methodology pioneered by QGEM 2011 (Margie et. al. 2013), worms were placed in a common starting area and their movement in response to a control substance and to the test odorant was observed. With an anaesthetic at target sites to immobilize worms at the end of each trial, the number of worms in the test and control quadrants were counted to calculate a chemotactic index. More details on how this assay works can be outlined here.
Isovaleric acid solutions at 1M, 7:1, and 99% concentrations were used. A total of 38 chemotaxis assay plates were run.
The above shows our results from the standard graph for our chemotaxis assay. According to statistical analysis, there was no significant difference between C. elegans motility response to the control and test samples.
In order to test the effectiveness of our part, it was necessary to create an assay to test our completed part against. Using the assay methodology pioneered by QGEM 2011 (Margie et. al. 2013), worms were placed in a common starting area and their movement in response to a control substance and to the test odorant was observed. With an anaesthetic at target sites to immobilize worms at the end of each trial, the number of worms in the test and control quadrants were counted to calculate a chemotactic index. More details on how this assay works can be outlined here.
Isovaleric acid solutions at 1M, 7:1, and 99% concentrations were used. A total of 38 chemotaxis assay plates were run.
The above shows our results from the standard graph for our chemotaxis assay. According to statistical analysis, there was no significant difference between C. elegans motility response to the control and test samples.
A huge thanks to our sponsors:
