Welcome to the engineering page!. Here we describe the engineering flow, the iterations, the successes, and the failures, and how, as we developed, conducted in-depth research, created, and modelled, we encountered difficulties that required many iterations and adjustments. You will see in this section that our project's development was not straightforward, making it worthwhile to highlight.
The design process to come up with our part sequence was an iterative one. During the design stage of our project, we explored and evaluated leading to a refinement in our designs and reworked in response to the problems that emerged as the analysis was conducted with our design.
🤔 Why Arsenic is an Ideal Element to be Detected.
An international team of researchers have found that when some soil minerals are enriched with arsenic (e.g. pyrite), gold can enter the mineral structural sites by directly binding to arsenic (forming, chemically speaking, Au(2+) and As(1-) bonds, which allows gold to be stabilized in the mineral. However, when the arsenic concentration is low, gold doesn't enter the mineral structure but only forms weak gold-sulfur bonds with the mineral surface [2]. This implies a relationship between the presence of arsenic and gold. Put another way, a patch of land with high arsenic concentrations is more likely to harbor short-term gold.
Per data collected in the Birimian of southwestern Ghana, where significant gold deposits have been found, arsenic concentrations of up to 40-58 ppm and Au concentrations of 200-260 ppm have been detected in prospective gold areas. In addition, it was found that pathfinder components suited for characterizing Au mineralization in intricate regolith settings were found to be As and Zn[3]. Soils overlying sulfide ore deposits commonly contain arsenic at several hundred parts per million; the reported maximum is 8,000 ppm. This arsenic may be present in unweathered sulfide minerals or an inorganic anion state [4].
Arsenic exists in the environment in two forms: organic and inorganic arsenic species. However, inorganic arsenic species, which include arsenite and arsenate, are the most prevalent forms of arsenic in the environment [5]. It should be noted that some of these sulfides, such as pyrite, with inorganic arsenic, come together to accumulate gold. From these findings and evidence, we can engineer the bacteria to detect arsenite (As (III), an inorganic form of arsenic).
🤔 Why Iron is an Ideal Element to be Detected.
Our initial design didn't include Iron as a pathfinder; however, to increase the chance of getting gold in the explored land using our biosensor, we discovered Iron as another potential pathfinder and included it in our design.
Instead of destroying E. coli, iron (Fe) causes two intriguing things to happen to it. Firstly, iron stimulates growth as it is an essential metabolite not available to E. coli in serum. Secondly, it interferes with bacterial killing, a process usually mediated by natural antibodies and complements [7]. However, the bacteria limit the iron pathogens can access during infection by generating iron- and siderophore-chelating proteins, exporting iron from intracellular compartments containing infections, and regulating dietary iron absorption. A transcription factor called Ferric Uptake Regulator (Fur) uses Fe2+ , as a corepressor. It inhibits the uptake of iron in pathogens [8].
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. However, when Fe2+ is absent, the mRNA is allowed to transcribe. Although FUR acts as a transcriptional repressor, it can be turned into an activator by preventing or eliminating another transcriptional repressor's ability to bind to DNA [8].
This design utilized an inducible Au promoter triggered in the presence of gold ions (Au+, for transcription, eventually causing the eforRed reporter protein to express a pink colouration (figure 1).
Figure 1a: Initial schematic of the Au sensing module based on the inducible promoter
With the idea of utilizing an inducible promoter, thus, the promoter comes on or becomes active in the cell in response to specific stimuli. They stay in an inactive stage unless they receive stimuli [11]. This served as the basis for our initial design (Fig. 2). The activator protein binds to the promoter in the presence of arsenite (As3+) and activates it to start transcription. This causes the reporter gene (amilGFP) to signal the transcriptional activity in the bacteria by causing the bacteria to glow.
Figure 2a: Initial schematic of the As sending module based on the inducible promoter
Biosensor systems have certain shortcomings, such as high leakage, low induced fold change and poor sensitivity. Leakages in genetic circuits exacerbate false positives. This is primarily true with linear designs. However, we need to use “secure” circuits that reduce leakages, hence, false positives. Typically, false positives would lead to an ineffective biosensor that will further worsen the problem we seek to address by giving false indications of areas that may not have gold [1]. Thus, this will increase pits and failed extractions. Our initial designs had limited control and operated in a direct "input-output” paradigm.
Also, in the initial designs, we utilized Au and As pathfinders. Although these trace elements were good pathfinders for gold prospecting, to increase the probability of the presence of gold after prospecting, we included another vital pathfinder for gold, thus, Iron (Fe) oxides. Studies in identifying pathfinder elements for gold in some areas of Ghana revealed that Fe is strongly associated with gold. Hence, the alternate and final designs incorporated the detection of Fe2+.
Figure 1b: Schematic of the sensing module for Au based on the PgolTS-golS-PgolB-amilCP regulatory circuit in the presence of Au
Figure 2b: Schematic of the sensing module for As based on the arsR operon which regulates As+
Figure 3: Schematic of the sensing module for Fe based on the FUR-PAceB-TetR-Ptet module in the presence of Fe2+
The evolution of the development of the bio-stake design within which the hydrogels(more about this in the safety section) are placed is highlighted below:
Figure 4: Engineering Design Evolution