The 2018 iGEM NYU Abu Dhabi designed a BioBrick that includes a gene that can be targeted for species-specific detection of Vibrio cholera.
Part BBa_K2808000 was designed by including N-acetyl glucosamine-binding protein A (gbpA) gene for the detection of Vibrio cholera. The gbpA gene in Vibrio cholerae codes for the N-acetyl glucosamine-binding protein A (GbpA), which is a chitin-binding protein involved in the attachment of V. cholerae to environmental chitin surfaces and human intestinal cells. The gbpA gene has been found to be consistently present and highly conserved in V. cholerae. GbpA has been used as a target gene for the species-specific detection of V. cholerae O1, O139, Non-O1/Non-O139.
Instead of the 39 bp sequence found in CTX RstA gene that is used by BBa_K2299000, our part BBa_K2808000 uses gbpA gene for the detection of V. cholera. The advantages of using gbpA as a gene target compared to ctxA are various such as ctxA is involved in the production of the cholera toxin production which requires special safety precautions during bacterial transformation, lab experiments, shipping reagents, customs, etc. whereas the use of gbpA solves these problems as gbpA is not directly involved in the production of cholera toxin.
gbpA BioBrick double digestion reaction
Figure 1: Agarose gel electrophoresis showing double digestion of the gbpA BioBrick BBA_K2808000 (gbpA gene (1458 bp) inserted into the pSB1C3 backbone (2070 bp)) with EcoRI and PstI. (Lane 1) 500 bp DNA ladder (Bio-Rad); (Lane 2) gbpA BioBrick; (Lane 3) gbpA BioBrick digested with EcoRI and PstI.
BioBricks PCR amplification
Figure 1: Agarose gel electrophoresis showing PCR amplification of: gbpA BioBrick BBA_K2808000; hipO BBA_K2808001; invA BBA_K2808002; lmo0733 BBA_K2808003. (Lane 1) 500 bp DNA ladder (Bio-Rad); (Lane 2) gbpA BioBrick; (Lane 3) negative control for gbpA BioBrick; (Lane 4) hipO BioBrick; (Lane 5) negative control for hipO BioBrick; (Lane 6) invA BioBrick; (Lane 7) negative control for invA BioBrick; (Lane 8) lmo0733 BioBrick; (Lane 9) negative control for lmo0733 BioBrick. Lanes 2, 4, 6, and 8 show bands around 1019 bp, 203 bp, 818 bp, and 403 bp respectively as expected for the PCR products of the gbpA, hipO, invA, and lmo0733 BioBricks respectively.
Special Track:
The NYUAD iGEM 2018 team immensely improved the project of the NYUAD teams of 2016 and 2017. We not only developed the existing process but also extended it to beyond the single pathogen the team of 2017 was working with.
The 2017 team worked on a portable device that could identify Shiga Toxin producing E.Coli over 30 minutes. This device utilised LAMP for amplification and the reaction took place in a gel with wells. The issues with this device are as follows:
1. User Interaction: The user was required to mix the food sample in a test tube and pipette it into each well separately. It can be noticed that this device was not user friendly as the target group were roadside food vendors. Most food vendors will not have the time or knowledge to use a pipette to check the food before serving it.
2. Variety of Pathogens: As mentioned earlier the device was able to find only one food pathogen and did not work have the choice to amplify other pathogens.
3. Device design: The device was not designed to suit the innate rush in a food stall/restaurant and did not take into account the different environments it might be used it. The device was also bulky and space within it was not optimised.
4. Method used: The team used LAMP for DNA amplification and it was visualised by fluorescence. This visualisation also needed a UV lamp, which is not cheap or easily available for the target group in mind.
While our team was brainstorming for new project ideas, we noticed these issues in EcoLamp and considered the option of improving it. This thought was further strengthened by our interaction with potential target customers and users. The team took each aspect from the above observations and improved them in the following ways:
1. User Interaction: The team worked on developing a hardware component that will include all the processes of sample collection and processing into one device. This meant replacing the test tube, cotton swab and pipette with one device, which was also contamination free and does not leak. The team after multiple tests, developed the sample collector which uses the ‘swab and press’ mechanism for sample collection. The sample is then added to the testing unit (chip) by the lid with a dripper. These additions simplify the numerous processes, thus making it suitable for any user with any background.
2. Variety of Pathogens: The biologists in the team worked hard and developed the existing protocol to suit multiplexing, which can be used in other devices made by Pathogene in the future. The team also worked on primers for four different pathogens which has enabled us to develop a device that can test for four different pathogens with one sample.
3. Device Design: Since Pathogene offers a series of customizable pathogen detection devices, it gives the user the option to choose a device depending on the environment. The device completed now, is suited for different kinds of travellers or people on the go, as it is handy and can be used with much ease. At the same time, it also takes into account people using it from a stationary place, thus giving the choice between battery pack and wire.
4. Methods Used: The team worked on both Lamp and RPA for the four different pathogens. This gives the user the option to choose high specificity with heating or comparatively less specificity without the additional heating. For visualisation, the team tested out fluorescence and colorimetry for both methods, thus adding more choices for customisation. The team also found that fluorescence can be visualised with specific kind of blue LED and incorporated that in the device design- thus replacing the UV light and making the overall product and process more accessible to everyone.
