Team:Bristol/Design
Here, you can read about how we designed our fragments for Gibson assembly, how we designed our primers, and our fuel cell design.
Fragment design
We were told by IDT that neither the Nap nor Nrf operon could be synthesised in one piece, as at around 8 kb each they were too long. Each operon was therefore split into 4 fragments of < 2kb. These fragments were designed with overlapping sequences of approximately 40 bp, to allow subsequent Gibson assembly. This is shown below (the same was done for both Nrf and Nap).
We successfully used PCR to amplify each fragment for both operons, however Gibson assembly proved difficult (see Experiments for more detail).
Primer design
We planned to submit Nrf and Nap as one composite part, as well as individually (see Parts). In order to do this, we designed two different forward primers for the first of the four Nap fragments (fragment "Nap1"). The first primer annealed to the very start of the Nap1 fragment and contained an overhang allowing homology with the end of fragment Nrf4. We refer to this new fragment created after PCR as "Nap1H" (Nap1 homology). Subsequent Gibson assembly using this new fragment would result in the composite part shown below.
The second primer annealed further along the Nap1 fragment, removing the double terminator which would not be required if Nap was used without Nrf preceding it. This primer had an overhang which contained the BioBrick prefix, allowing Nap to be used as a part on its own. We refer to this fragment as "Nap1-dt" (Nap1 minus the double terminator). Use of this fragment in Gibson assembly with Nap2, Nap3 and Nap4 would result in the following part:
The primers we used are shown in the following table. The portions shown in blue are overhangs and do not anneal to the initial fragment to be amplified.
Primers for fragment submission
As the failures with Gibson assembly prevented us from assembling the whole operons within the timescale of the project, we designed new primers that would allow to isolate and submit whole coding regions from our fragments. Specifically, we designed primers to isolate NrfB from Nrf2, NrfD from Nrf3, NrfF and NrfG from Nrf4, NapF and NapD from Nap1, and NapB and NapC from Nap4 (shown below). These primers bound at the very beginning and end of the coding sequences, and introduced prefixes and suffixes where needed to convert them all to BioBrick parts.
Pod design requirements
Our pods must be able to:
Consume NOx pollutants.
Be a feasible solution to removing pollution.
Produce a useful product.
Function and be sustainable.
Solutions:
We chose to upregulate NrfA (ccNir) which reduces nitrite (a NOx pollutant) to ammonia. This enzyme already exists in E. coli, and therefore expression should be easily compatible.
The incorporation of the Nap operon, which reduces nitrate to nitrite, will increase the effective input of NOx into NrfA, as NrfA cannot directly consume nitrate.
The ammonia produced by NrfA is in itself a useful product, but could potentially be fed into a fuel cell to produce electricity. The electricity produced could be used in some way for maintenance of the pod.
Fuel cell design
To think about the future value and sustainability of our project we felt it was important to consider possible uses for the ammonia produced. Although ammonia itself is a useful by product and is used in fertilizers, cleaning products and industrial processes, harvesting the ammonia for these purposes would increase costs and most would lead to ammonia being fed back into the nitrogen cycle in the form of a nitrogen compound thus replenishing the nitrogen oxides (NOx) we are aiming to reduce. Therefore we took inspiration from the literature, and gained valuable insight from the Bristol Robotics Lab (BRL), to design a system in which the ammonia produced could be used to generate electricity.
This involves feeding ammonia into a fuel cell; there are multiple examples of ammonia being used within a fuel cell. However, most of these are within solid oxide fuel cells (SOFCs) which run at temperatures too high for our bacteria to survive. Bacteria would therefore need to be kept in separate chamber; this would increase both the cost of production, and the complexity of the design. SOFCs also present other problems such as start-up times and brittleness of components which are often made of ceramics. Traditional proton exchange membrane fuel cells (PEMFCs) are incompatible with using ammonia directly as a fuel due to the acidity of the membrane. Two options reported for using ammonia in PEMFCs are to convert the ammonia to hydrogen and nitrogen using either high temperatures or electrolysis, then using the hydrogen produced as fuel. Clearly this would be impractical as either of these would consume energy making the system less efficient as well as increasing cost.
We therefore chose to use a system based on an alkaline anion exchange membrane (Fig. 1) in which the bacteria could hopefully sit within the anodic chamber producing ammonia which could be directly oxidised with the aid of an OH- group at the anode, made of chromium decorated nickel (97.7:2.3). The cathode, made of manganese dioxide (MnO2) (20% wt MnO2) would be exposed to air; in the presence of water this would result in the reduction of oxygen, producing the OH- group which would subsequently be transported across the membrane to react with ammonia at the anode. The resulting products would be water and nitrogen from the anodic chamber, water and air produced in the cathodic chamber, and current resulting from the oxidation of ammonia. We chose the materials for our anode and cathode based on a paper written by Lan Rong and colleagues, who used these as substitutes for the traditional materials such as platinum to reduce cost.
Fig. 1 - Diagram illustrating functional flow of an ammonia fuelled alkaline anion exchange membrane fuel cell. Rong Lan, and Shanwen Tao Electrochem. Solid-State Lett. 2010;13:B83-B86
The outer casing will be made from acrylic panels, which can be autoclaved. These are each separated by a gasket and held together using plastic bolts. The anodic and cathodic chambers will be separated by the anion exchange membrane and have an internal volume of 1400mm³ (10x70x70mm). The external construct was provided by the Bristol Robotics Lab (BRL) and from this we designed our own model of the MFC so we could create more if required. The two major benefits of using the BRL's design are:
Quick and easy to manufacture/build to allow for fast construction.
Made from cheap, easy to source materials due to a tight budget.
To capture the NOx and dissolve it in solution we aim to use a dry deposition and washing system which will feed into the MFC. One problem we have encountered whilst devising a solution to the capture and isolation of NOx is the separation of nitrogen and sulfur compounds as both elements are likely to stick onto our capture surface. Sulfur is undesirable as this may result in unwanted reactions within the fuel cell and have detrimental effects on the survival and metabolism of E. coli.
Upon completing this MFC design with sufficient power output using our recombinant E. coli the next step would be to combine individual units in an array connected in series-parallel to increase power output. This array would be contained within a ‘pod’,
the design and placement of which will be determined alongside significant input from the general public and public officials.
