We contacted stakeholders who work on biofuel production in both industrial and academic positions. In the end, stakeholders who worked in universities provided us with much more detailed feedback and requests for our project design, so we have chosen to integrate our human practices to the feedback from three stakeholders in various universities.
Nathan Tague Ph.D. Candidate in Biomedical Engineering at Boston University
Advice: Nathan gave us some very useful feedback for the implementation of our scheme. He works on improving biofuel synthesis in the Dunlop Lab. Nathan agreed that building our model to export alkanes by introducing channel protein would simplify purification , but he also importantly noted that “continuous production will result in many more divisions, and potential drift or non producing escapers taking over the population if your production scheme has a fitness disadvantage. The more components you add and the more sophisticated the genetic circuits get, there are that many more nodes for the system to mutate and not produce.” When thinking about our long term goal of developing cost effective method to produce these biofuels will have to keep this in mind. Further, Nathan suggested that looking forward we should check into the specific promiscuity of the thioesterase of choice. Thioesterase usually make a mix of FFA of varying chain lengths; therefore, enzyme engineering or balancing certain FAS components would ultimately allow us to favor certain chain lengths.
Integration:Nathan's advice, challenged us to understand the design constrains of our organism. With his input, we decided to design a system to minimize the metabolic pressure that we place on our cyanobacteria. It is for this reason that we decided to regulate the production of the fatty acids during the day when the cell has the greatest metabolic capacity while producing the alkanes at night when the cell’s metabolic demand is lowest. Through this method, we hope to minimize the selection against our central constructs.
To minimize the loss of our plasmids between generations, thereby reducing genetic drift, we designed the parB system to ensure faithful segregation of the low copy number plasmids in our system during replication. As this is a host mechanism for stable plasmid is within the organism, we hope this system will help us minimize genetic drift and allow our system to function during the growing season.
Laurens Mets, Ph.D. Associate Professor, Molecular Genetics and Cell Biology and Founder of Electrochaea
Advice: Professor Mets played a critical role in the development stage of our iGEM project this year. Dr. Mets works on applying genetic engineering to the improvement of photosynthetic production of hydrogen from water as an environmentally friendly fuel. While he recognized alkanes a viable fuel product, he advised us to develop an export system to limit the toxic effect of the alkanes and improve the efficiency of the harvesting process. Additionally, Dr. Mets, given our concern with photosynthetic inefficiency, suggested that it we identify a way to optimize the way our system produce the fatty acids.
Integration: Professor Mets' advice lead us to develop a mathematical model to ensure that we bias our cells for fatty acid synthesis production. With his help, we deiced to create the Fatty Acid Synthase Operon so that we could better modulate the production of our desired products. Furthermore, the entire inspiration for the Exporter operon comes from Professor Mets' concern that alkane toxicity would limit production and concerns for inefficient harvesting. Michael Disare PhD Candidate in Chemical Biology at University of Chicago Advice:Michael recommended to us that since fatty acid synthesis is over multiple different enzymes, we can modulate the expression at many different places. He recommended to us that one strategy we could use to make our construct more efficient was to ensure that we are expressing our fatty acids synthase enzymes in a balanced fashion so that we are not wasting protein while ensuring maximal product production.
Integration: Michael suggestion of trying to optimize our fatty acid synthesis operon is the reason for our mathematical model. The mathematical model provides us insights on the optimal ratio of the enzymes to ensure the highest fatty acid synthesis while minimizing energy use through protein anabolism.