Discovering past projects
During our initial research phase, we also looked through what other iGEM teams have done in aiding bowel cancer detection, particularly via use of biosensors, in order to get inspiration on how we should approach the problem. The one that caught our attention most was UPF-CRG Barcelona's 2016 project - Polybiome - which, in addition to the probiotic that reduces the amount of polyamines absorbed by gut cells, they also engineered a visual system for detection of polyamines. What struck us was the similarity in concept between our project and theirs - both involved biosensors used in detection of a telltale marker of developing bowel cancer.
The idea that polyamines, essential for cell growth and development, could become carcinogenic when present in high enough concentration, stood out to us due to the parallels that could be drawn between the molecular level of life and the behaviours of humans. Looking through past work in elucidating how cells metabolise polyamines, a crucial process due to their involvement in production of coenzyme A, we found that there was much we could add to aid future iGEM teams in exploiting the link between high polyamine concentrations and occurrence of bowel cancer, in hope that this property could be combined with other testing kits, including our own, to create an all-encompassing testing kit for a wide range of bowel cancer markers.
Research and characterisation
Our research into UPF-CRG Barcelona's 2016 project led us toFMS1 - a polyamine oxidase isolated from Saccharomyces cerevisiae, which degrades polyamines to smaller chain amines. Following informations from the papers referenced below, we decided to focus on FMS1 due to its close ties with our intended research, and although we were unable to characterise the part experimentally in a lab setting, we were still able to find vital information which helped us build on our project, and hopefully projects yet to come.
In vivo, it is an important precursor in pantothenic acid biosynthesis, and hence in coenzyme A production. This enzyme is therefore crucial to the cell's energy metabolism.
FMS1 has been found to use FAD as a cofactor, using it as the oxidising agent in a redox system. The enzyme operates at maximum efficiency in an acidic environment (ph ~ 9), which suggests that some of the amino groups of the substrate have to be protonated for proper binding - indeed, it has been suggested by the three-dimensional model of FMS1 that asparagine residues in the binding pocket stabilise protonated amino groups. FAD has been found to be reduced in an irreversible step, in which the substrate is also oxidised. From there, the product can either disengage from the reduced enzyme-product complex, or the whole complex can be reoxidised, after which the product disengages from the oxidised enzyme-FAD complex, restoring the enzyme and the cofactor. The rate constant for both release reactions are much lower than the rate constant for oxidisation of the substrate (4,5 s-1 and 56,5 s-1, respectively, as opposed to 126 s-1), therefore the RDS of the whole reaction is the product release step.
Read more about this on the Parts Registry.
- Landry, J., Sternglanz, R., Yeast Fms1 is a FAD-utilising polyamine oxidase, Biochemical and Biophysical Research Communications, 2003, 303, 771-776, doi: https://doi.org/10.1016/S0006-291X(03)00416-9
- Adachi, M. S., Torres, J. M., Fitzpatrick, P. F., Mechanistic Studies of the Yeast Polyamine Oxidase Fms1: Kinetic Mechanism, Substrate Specificity, and pH dependence, Biochemistry, 2010, 49, 10440-10449, doi: 10.1021/bi1016099