Team:UCC Ireland/Biosensors
Synthetic biology allows for the creation of biological sensors from preexisting natural genetic circuits. Since biosensors can link a multitude of sensor and transducer elements to a wide range of readouts, they have huge potential for use in many different industries worldwide. They can be used in both in vivo and in vitro to measure the concentration of the desired analyte. Biosensors can be used in diagnostic tests and can even be utilised in agriculture to measure the fruit ripening process (iGEM team Sydney 2016) and can detect environmental hazards (iGEM team Ionis). Biological sensors incorporate biological components in either one or both of the “sensor” and “transducer” elements. When the analyte binds downstream genes are transcribed including reporter read-out genes that allow for quantification of the analyte.
Having a simple standardized colorimetric readout that quantifies the concentration of drugs and contaminants, may be very useful to ensure consumer safety in the food and beverage industry. Currently, all processed food and beverages are subject to rigorous testing for contaminants that are hazardous to human health. However, this testing can be expensive, inaccessible and time-consuming. Since these sophisticated tests are often limited to large-scale producers, local dairy farmers, microbreweries and home-brewers remain vulnerable to penalties and poisoning should the level of contaminants in their products fall outside the regulatory guidelines. Our iGEM project aims to create an affordable, easy to use and reliable biosensor, that will be incorporated into a portable colourimetric device, to detect antibiotic residues in milk and methanol in alcohol. A cell-free system will be utilised to circumvent the risks associated with the use of genetically-modified live bacteria outside the laboratory.
Overview
The Erythromycin and Methanol biosensor were first cloned into plasmids, and transformed into in live Dh5 cells to test their functionality. Characteristics of each gene circuit have to be assessed and optimised to ensure that the final consumer product is reliable and useful. In general, Moonshine biosensors aim to signal the presence of illegal amounts of antibiotics in raw farmyard milk, based on legal and governmental policies, and to warn of toxic levels of methanol in alcoholic beverages, based on medical recommendations and legal limits within a short period of time. Therefore, the following issues need to be taken into account in optimising these constructs for real-world application: specificity, sensitivity and response time.Erythromycin Biosensor
Given that the concerns surrounding the use of genetically-modified organisms in farmyard environments are manifold and rather nebulous, we considered incorporating our genetically modified biosensor constructs, especially the Erythromycin sensor, into a cell-free system. We have developed a slider device (that can be attached to any ubiquitous smartphone) along with an app, which can correlate the level of fluorescent protein expressed in a cell-free solution in a PCR tube (that is inserted into the device!) to the concentration of erythromycin present.Specificity:
The mph(A) promoter is depressed in the presence of a large range of macrolide antibiotics. While the system is not induced by other classes of antibiotics such as tetracyclines, aminoglycosides and penicillins, it responds to 12, 14 and 15-membered macrolides and not only 14-membered, erythromycin in particular. This postulates that there is a certain degree of flexibility of the sensor in detecting core structural components of macrolide antibiotics. (Möhrle, Stadler and Eberz, 2007). Hence, 16-membered clinical veterinary antibiotics such as spiramycin, josamycin, tylosin, and tilmicosin may promote transcription of the readout gene as well. (Veterinary Manual, 2017) While this would promote widespread usage of our biosensor, it raises concerns about whether this flexibility would cause our biosensor to respond to other chemicals with similar structural components.Sensitivity:
The maximal residual limit for erythromycin in milk in the EU is 40 μ g/l of raw milk. This is an extremely small concentration. We have noted that the pJKR-H-mphR is inducible at concentrations as low as 1 μM (which corresponds to approximately 736 μ g/l). As such, further optimisation and more rigorous testing with smaller concentrations of the inducer: erythromycin to characterise its behaviour will be required. In the future, we suspect that the ProB constitutive promoter may have to be replaced with a weaker constitutive promoter to stunt production of the mphR(A) repressor. This may allow lower concentrations of erythromycin to inhibit the repressor binding to the mph(A) promoter region. However, a fine balance needs to be achieved. Having an extremely weak promoter for mphR(A) might result in lack of repression of mph(A), and cause constitutive expression of sfGFP and AmilCP. Again, we don’t want that because it might jeopardise the reliability of our system by producing false positive results.Response time:
Our results show detectable levels of green fluorescent protein production by the pJKR-H-mphR plasmid within 0.5 hours of induction. This indicates that our sensor has the potential to be just as fast, or even faster, than the SNAP test that is currently available on the market. The SNAP test claims to be able to detect levels of antibiotics within 6 minutes. Further testing of our sensor within smaller time intervals will be necessary to ascertain whether modifications need to be made in our biosensor for it to compete with this rival product in the market.Methanol Sensor
Specificity:
Due to the structural similarity between methanol and ethanol which would invariably also be present in the alcoholic beverages that our sensor would be used to test, we need to determine specificity for the methanol dehydrogenase enzyme for ethanol and methanol, and also observe the degree of binding of ethanal compared formaldehyde for the repressor protein EcFmrR, to determine if the presence of ethanol has an impact on the fluorescent readout of our sensor. This can be achieved by assaying the sensor in the presence of methanol, with ethanol and with a combination of both.
Sensitivity:
Although not difficult to qualitatively determine the presence of methanol using our sensor, further testing is also required to ensure that the receptor is sensitive enough to detect the allowable levels described by legislation for alcoholic beverages which is as low as 0.01%w/v for spirits like vodka. (European Commission) Assays will be performed to determine the minimum concentration detectable by our sensor to determine it’s suitability for usage in different beverages.
Feasibility & usefulness:
One difficulty that could arise, is the ability of the sensor to detect a wide range of methanol concentrations depending on the alcoholic beverage which varies from 1% w/v for beer to 0.01% w/v for spirits like vodka. Possible sensor modifications could be made to the the sensor however to accommodate this, like varying the promoter of the the methanol dehydrogenase gene which is currently J23101 or by altering terminators, to vary the degree of ‘Leakiness’ of transcription of genes.
Sensitivity:
Although not difficult to qualitatively determine the presence of methanol using our sensor, further testing is also required to ensure that the receptor is sensitive enough to detect the allowable levels described by legislation for alcoholic beverages which is as low as 0.01%w/v for spirits like vodka. (European Commission) Assays will be performed to determine the minimum concentration detectable by our sensor to determine it’s suitability for usage in different beverages.Feasibility & usefulness:
One difficulty that could arise, is the ability of the sensor to detect a wide range of methanol concentrations depending on the alcoholic beverage which varies from 1% w/v for beer to 0.01% w/v for spirits like vodka. Possible sensor modifications could be made to the the sensor however to accommodate this, like varying the promoter of the the methanol dehydrogenase gene which is currently J23101 or by altering terminators, to vary the degree of ‘Leakiness’ of transcription of genes.
