What can be done to take our work further
Our improvements to the efficiency of photosynthetic fuel cells have been significant if unspectacular. Synechocystis is not commonly used in synthetic biology and, lacking substantial help from a university, we spent much of our time tackling the difficulties associated with successfully modifying it. This has helped us to grasp some of the problems with photosynthetic fuel cells and genetic modification of Synechocystis. In turn, this has suggested several paths for future research and experimentation.
First one ought to address the question of what might be done to make modifying Synechocystis a little easier. At the moment, there are very few BioBrick-comaptible integrative plasmids that are suitable for use in Synechocystis and cyanobacteria more generally. This meant that we were forced to use replicative plasmids. The first step in any future project would be to develop BioBrick compatible plasmids. There is also a lack of promoters and ribsosome binding sites – as we found out to our cost.
Secondly, it is important to ask what can be done to improve the efficiency of photosynthesis in Synechocystis. Presently, the limiting factor appears to be the efficiency of its carbon-concentrating machinery – specifically Rubisco. However, altering Rubisco’s activity does not appear to have a significant effect on photosynthetic efficiency or growth rate . Instead, it seems more prudent to focus on the other components of the carbon-concentrating machinery. One could solve this problem in one of two ways. First, one could attempt to increase carbon dioxide concentration around Rubisco. This was the approach that we decided on. Second, one might attempt to cut out Rubisco entirely and re-engineer the photosynthetic pathways in Synechocystis. However, this would be a difficult endeavor (to say the least) and is hardly the most practical solution to the problem at hand. Instead, we recommend that future efforts should be concentrated on designing effective ways to increase the carbon dioxide concentration around Rubsico by increasing the efficiency of Synechocystis' existing carbon-concentrating machinery.
Third, one ought to consider the practical problems with exporting electrons out of the cell. Recent studies have shown that electron export is the primary limiting factor on the efficiency of BPVs . It is all very well to have a cyanobacteria that can photosynthesise efficiently. But a fully-functioning fuel cell must be able to harness the electrons produced by photosynthesis. In many respects, this is the difficult part. Electrons are exported from synechocystis without the addition of external mediators . However, the rate of electron transport is low and mediators are usually required. There are three main classes of electron carriers: phenazines, flavins and quinones. It is unknown whether or not any of these are involved in electron transfer in Synechocystis - this would be fertile ground for future research. Synechocystis lacks the ability to synthesise phenazines, so they are unlikely to be of much use. By contrast, it can synthesize flavins and quinones fairly easy and so it seems sensible to concentrate on these two classes of electron carrier. It should be noted flavins are non-lipid soluble and so can only exit the cell via porins . Thus it would be necessary to over-express the genes that code for porins as well as those involved in flavin synthesis. However, the use of mediators is impractical on a large scale. Ultimately, it is likely that any satisfactory solution will involve the re-engineering of metabolic pathways so that electron flow is directed out of the cell.
Fourth, it should be noted that Synechocystis is probably not the ideal organism for use in fuel cells since it has a long generation time and is hexaploid. Synechocystis does have its advantages: its genome is well understood and it is easy to transform as it readily takes up plasmids by natural transformation. But if fuel cells were ever to be commercially viable then another cyanobacteria ought to be found. Of course, one might ask why cyanobacteria should be used at all. What about photosynthetic eukaryotes? In fact, there is a very simple reason for using cyanobacteria rather than another class of organism – cyanobacteria, as prokaryotes, have far fewer membranes than any eukaryotes. This reduces the number of electron transfer steps across membranes required . As such, cyanobacteria are the natural candidates for use in BPVs.
This article has briefly explored several areas that might be researched more fully by iGEM teams to come. BPVs are an exciting area of research and we hope that some other team will choose to focus on them. In that case, our project may act as a source of ideas and a guide to the problems involved in modifying Synechocystis and developing more efficient fuel cells.
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