Pakistani iGEM team’s biosensor detects vehicle emission levels

Air pollution from exhaust fumes is a major problem in the developing world

More than 80% of the global urban population is exposed to air that does not meet the quality standards provided by the World Health Organization (WHO). In developing countries, air pollution levels exceed WHO limits in 98% of cities. As a result, air pollution causes an estimated 5.5 million premature deaths worldwide—more than malnutrition, obesity, and alcohol and drug abuse [1].

Carbon emission is by far the leader of all greenhouse gas emissions, and transportation is among the highest contributors to carbon emissions. Two dangerous constituents of vehicle exhaust fumes are carbon monoxide (CO) and nitrogen oxides (NO_x). Pakistan contains 3 of the 20 most polluted cities in the world and has been listed by the World Bank as suffering from the most profound air pollution in South Asia [2]. However, for Pakistan and many other developing countries, vehicle emission regulations are either not present, or not properly implemented or enforced.

Lack of regulation enforcement in low-income countries is partly due to the absence of expensive, non portable instruments required to detect vehicle exhaust emissions. These include Non-Dispersive Infrared (NDIR) and Gas Chromatography (GC) for CO, and Gas Phase Chemiluminescence for NO_x detection. Therefore, what many of these communities need is a method that is portable, easy-to-use, and inexpensive, thus making it accessible to local government agencies.

Figure 1. Significance of exhaust pollution and a model for simple detection. NDIR = non-dispersive infrared, PPM = parts per million. (Figure courtesy of 2016 iGEM Team Peshawar.)

Replacing expensive detection methods with a simple, portable test

With the goal of developing a biological device that could cheaply measure vehicle pollutants, 12 undergraduates from across Pakistan gathered in Peshawar during the summer of 2016 to form Pakistan’s first iGEM team, Team Peshawar. The group envisioned a bacterial biosensor that could detect CO and NO_x levels in vehicle exhaust and provide colorimetric readouts through chromoprotein expression.

The Peshawar team designed a system of 2 separate but coordinated gas-sensing genetic circuits. The CO-sensing circuit uses the CooA transcription factor and 2 CooA-dependent promoters, CooF and CooM. The NO_x-sensing circuit uses the NsrR repressed promoters, YeaR and NirK, as well as the NorV promoter.

After successful modeling and simulation of their concept, they genetically engineered E. coli cells to incorporate both the CO- and NO_x-detecting circuits. In the presence of external CO, the CO-sensing circuit is activated and produces a blue color. This occurs when 2 molecules of CO bind the CooA transcription factor to form a CO–CooA complex, which activates the pCooF promoter and results in transcription of a gene that expresses blue chromoprotein.

Likewise, external NO_x activates the NO_x-detecting circuit. Molecules of NO_x attach to the NsrR repressor, activating the pYeaR and pNirK promoters (p). The promoters in turn transcribe a gene expressing a yellow chromoprotein. When both CO and NO_x gases are present, the cells express both blue and yellow chromoproteins, producing a shade of green (Figure 2).

Figure 2. Design of an E. coli “biosensor” that expresses chromoproteins in the presence of CO and NO_x gases. This model of the 2016 iGEM Team Peshawar engineered E. coli cell depicts the CO- and NO_x-detecting constructs. The presence of CO activates the CooA transcription factor, which triggers activity of the CooF promoter, pCooF, resulting in expression of a blue chromoprotein. Likewise, the presence of NO_x inactivates the NsrR repressor, releasing the promoters, pYeaR and pNirk, to express a yellow chromoprotein. When both gases are present, the cell will express both blue and yellow chromoproteins, resulting in a green color. (Figure courtesy of 2016 iGEM Team Peshawar.)

gBlocks Gene Fragments crucial to project success

The CO- and NO_x-detecting systems in Team Peshawar’s project required 6 different DNA constructs, which ranged from 1500–6300 bp. Each contained 4 genetic elements (a promoter, a ribosomal binding site, a protein coding region, and double terminators). IDT donated 20 kb of custom, synthetic dsDNA fragments, in the form of gBlocks Gene Fragments, that greatly simplified building these expression systems.

"Given the rarity of synthetic biology research in Pakistan, we were probably the first to use gBlocks Gene Fragment synthesis,” shared Team Leader, Muhammad Ali. “Continued correspondence with IDT personnel provided valuable guidance—from the ordering procedure to cloning protocols—which really helped us execute our project. The gBlocks Gene Fragments arrived in only a few days, a pleasing result in a country where primers usually take weeks to months to reach us. We are proud to have initiated the synthetic biology legacy in Pakistan through our iGEM project, and we have IDT to credit for making it possible.”

Figure 3. 20 kb of synthetic dsDNA fragments (gBlocks Gene Fragments) donated by IDT to Team Peshawar. These custom dsDNA fragments greatly simplified building Team Peshawar’s expression circuits. In 2016, 250 iGEM teams took advantage of IDT’s donation of gBlocks Gene Fragments. (Figure courtesy of 2016 iGEM Team Peshawar.)

A simple test strip for detecting exhaust fumes

Team Peshawar envisions their final product as a test strip that can be used in real world settings to detect CO and NO_x levels in vehicle exhaust fumes. The strip would contain a box with 2 sections: one with the engineered bacteria in nutrient media, and one with bactericides that would act as a “kill switch” to destroy the engineered cells. The strip could be used several times until the media is depleted.

While the E. coli strain used in this project is non-pathogenic and genetically modified such that it cannot colonize the mammalian gut, the kill switch provides control over horizontal gene transfer. The twister, when turned, would break the wall in between the sections, degrading the engineered bacteria (Figure 4). “In its final format, the team sees expanded uses of the test strip”, revealed Muhammed Ali. “It could be used not only for vehicle exhaust fume detection, but also for sensing CO leakages in indoor equipment, which can be fatal.”

Figure 4. Test strip within a kill switch design would detect CO and NO_x in vehicle exhaust fumes.

Team Peshawar advances cutting edge synthetic biology in Pakistan

The first Pakistani iGEM team included 4 women and 8 men from universities across the country, who spent an intensive 4 months developing and executing their prize-winning project (Figure 5). Team Peshawar was sponsored by the Directorate of Science and Technology, Khyber-Pakhtunkhwa, and was supported by the Institute of Integrative Biosciences at CECOS University. "We have been waiting for this moment to introduce synthetic biology in Pakistan, and what better way than to give students the opportunity to do cutting edge biotechnology research," said Dr Faisal Khan, Principal Investigator and Team Supervisor, who is also the director of the Institute of Integrative Biosciences at CECOS.

Figure 5. 2016 iGEM Team Peshawar. (Figure courtesy of 2016 iGEM Team Peshawar.)

In November 2016, the Pakastani undergraduate students competed with 300 teams in the 2016 iGEM International Jamboree competition, which included teams from top education institutions such as Harvard and Oxford. Their project won a bronze medal.

IDT sponsorship

For years, IDT has supported iGEM participants in their efforts to create solutions to all kinds environmental, medical, and technical problems. Support comes in the form of custom, high quality, double-stranded DNA fragments—gBlocks Gene Fragments. In 2016, over 250 teams took advantage of our offer to receive up to 20 kb of gBlocks Gene Fragments for free. Find out more at www.idtdna.com/iGEM.