In addition to doing a solid job, our aim was to lay the groundwork for future teams to expand on. Given the limited knowledge about synthetic biology on the continent, our contributions were also geared towards supporting the development of knowledge among diverse youth groups through outreach programs. We believed that creating tools that other iGEM teams can use to promote inclusivity as well as developing comprehensive guides for future outreach projects would help with this. In particular, we developed a novel iron biosensor, a guide for conducting fun synthetic biology sessions among young people, and a documentation for developing a multipurpose stake for biosensing purposes.
During the team’s initial research, we came upon fully developed sensing systems, both in literature and on the IGEM parts registry, that greatly expedited our design phase. Therefore, we intended to also create a useful resource for future teams with regard to biosensing. In particular, our most relevant contribution in terms of parts came from our creation of a novel iron sensing module that produces a pink color indicator, eforRed, upon sensing Fe2+.
The module we created consists of a constitutive promoter Pcat which induces the production of the FUR protein. In the presence of Ferrous ions, FUR acts as a repressor. In other words, it binds with the Fe2+ to undergo structural change. mRNA transcription is then inhibited when the homodimeric Fur-Fe2+ complex attaches to the DNA at a Fur binding site. When Fe2+ is absent, however, the mRNA is allowed to transcribe. In this case, the FUR protein acts as a repressor for the PAceB (since PAceB possesses an FUR binding site). In the absence of Fe2+, the PAceB is turned on, allowing for the transcription of TetR. This ensures that TetR binds and represses the Ptet, inhibiting further transcription downstream. Therefore the reporter gene, eforRed, is not expressed.
In the presence of Fe2+ , the FUR protein binds with the iron ions, repressing the PAceB. That way, the TetR protein is not transcribed. This allows Ptet to freely transcribe the reporter gene eforRed, giving out a pink glow. To the best of our knowledge, this is a new design and can easily be used in EColi for iron sensing purposes.
These protocols can be used by teachers or future iGEM teams for educational outreach or provide a practical feel of biology lessons taught in the classroom. The first and second activity focuses on DNA and its structure, and the third activity focuses on creating recombinant plasmids. Prior to the implementation of these practical activities, the tutors or instructors are advised to conduct brief lessons on the topics with the students to create the foundation for understanding.
Fig. 1 (A) Some materials for the Banana Extraction Activity, (B) Results after DNA Extraction in a test tube
Fig. 3 (A) & (B) Haribo candies used for building the helix structure of the DNA , (C) DNA structure built with the Haribos and toothpicks
Physical build of electronics and biosensor containment system
With the use of electronics, we were able to design our bio-electronic system. There were numerous stages involved to design the prototype.
Coding in C++: An Arduino code was used to order the microcontroller, to enable the color sensor to read the color indicators at set times, and display the results on an OLED screen. The code can be found on GitHub. The only library required is the “U8glib.h” zip file that can be found on GitHub as well. By calling this function, the user will be allowed to use the 1.3-inch OLED, will all its numerous capabilities: bitmap image drawing, scrolling of text, and switching pages of the screens. The next thing to be done is setting pins on the Arduino that can be used. Please note: Arduino can be used. We would encourage the use of the nano however, because it is more compact, and can be soldered on a perforated board. After setting pins for the two RGB’s, and the switching pin of the relay module, the void setup(), and the void loop(). The void setup() is where items are called once, for example the kind of pin that should be set:
pinMode(redPin,OUTPUT);
The void loop() is the part of the code where items are set to loop, this is where the relay module will switch on/off the U.V kill switch by replacing the wait time, after particles are detected. Furthermore, the OLED display printed text, can be reworded by altering the words in speech marks.
u8g.setFont(u8g_font_unifont);
u8g.drawStr(2, 20, "Fe2+ detected");
u8g.drawStr(2, 39, "Low Chance");
Arduino IDE coding of electronics
Soldering electronics:
It was imperative that after designing a working circuit on a breadboard, that the devices be properly fastened to a secure board. In so doing it will be a robust design that can be used in a real-world setting. For this step, 10cm x 10 cm perforated board should suffice – if the Arduino nano is used. One must be very careful not to let the wires routed to a specific path, touch other wires. This will prevent the on-board circuit from working at all. A soldering iron and a bit are required. Below is a circuit diagram of that will assist designers with connections. The circuito.io app was used to also estimate the price of each component used.
Electronic circuit and build-up
Building containment unit (CAD and physical design):
The PVC pipes were buckled in a neat wooden frame that made it easier to pour sand into the structure, to allow users to view and understand concepts, at close range. For the CAD model of the design, we have open sourced our CAD model for download on the GrabCAD digital library. The CAD model was made in three parts. The ball valve was drawn in Fusion 360 and assembled with the lower-end (11cm PVC pipe) and the longer (60cm PVC pipe). The lower-end was extruded and cut, to make 16 holes around the pipe. For the physical design, two ¾ inch pipes, caps, and brackets were bought and fastened to a 70cm x 35cm wooden board. A plexiglass front cover was also cut out using a CNC router – at Ashesi University. A larger 2 inch pipe was also used, and it served as a means to contain the stake. The wooden container was screwed together, and sand papered.
Designing the physical prototype and CAD model
Testing and feedback:
The final product test ensured that all electronics worked well, and that the prototype stake will be a good fit for users. We compiled the necessary feedback given to us by stakeholders that we presented our product to. The numerous stakeholder suggestions were what made our proof of concept more comprehensive and appropriate.