The most used, sugar requiring chassis in iGEM and molecular biology, Escherichia coli, is one of the tools our project, Merculess, used to build constructs. But for our primary chassis we wanted the CO2 utilizing photosynthetic model organism Synechocystis sp. PCC 6803. The model organism’s genome is known and many molecular engineering tools have been developed for it, but there is still a lot of research needed. We wanted to promote the research done on it because of the potential this light-driven chassis shows for different renewable applications that utilize CO2 as the primary carbon source. There is a global need for new sustainable biotechnologies and with Synechocystis we can fill this need and apply it to many industries.
Synechocystis has many special attributes, most importantly its photosynthetic
qualities. This means all the energy needed for growth and metabolism it gets from light and carbon
(CO2). This is a huge advantage because there are plenty of the required primary sources.
Synechocystis has been compared to E.Coli as being the model organism of photosynthesis. Therefore, there has been plenty of research done around the chassis. The research has given us the proper tools and protocols to work with. In addition to this, the Department of Molecular Biology at the University of Turku has been doing research on Synechocystis for years, so we had the proper infrastructure and knowhow at our hands for this project.
Our most important tools when working with Synechocystis are knowledge and
experience of building constructs with this organism. We had the required knowhow on genetic regulation
elements that allowed us to make reasoned decisions and well working constructs. On top of this Synechocystis is naturally competent so it has the inherent ability to take up and
incorporate foreign DNA into its genome without any special treatment that is usually required.
In our project, Merculess, we wanted to use Synechocystis as our chassis for many reasons. One reason was research done around mercury metabolism in the organism. It is known that Synechocystis has a native merA gene and mercury-regulators which we utilize (Singh et al., 2019). And most importantly because of its photosynthetic abilities it was the right chassis for us.
Our aim was to modify Synechocystis to convert extremely toxic methylmercury into
its less toxic elemental form. This would be accomplished by the overexpression of two enzymes closely related
to the conversion of methylmercury. We decided to achieve our aim by designing and optimizing a construct for
overexpressing both mercuric reductase (MerA) and alkylmercury lyase (MerB). Our genes of interest originated
from several different organisms (E. Coli, Synechocystis
and Pseudomonas Aeruginosa). The chosen genes could be expressed either individually
or together in an operon so that a single construct will express both genes. This allows us the possibility of
determining which genes work the most efficiently.
We built our constructs using a Golden Gate based assembly system that had recently been developed by the Department of Molecular Biology at the University of Turku and already had a library for us to use. This particular system enables the construction of expression plasmids in a single step assembly reaction. The assembly system has been developed specifically for the expression of heterologous genes and its purpose is to make it possible to easily optimize the translation of genes by allowing flexible variation of RBSs, which makes it the right choice for our needs of modularity. This approach enables the building of an efficient expression system of merA and merB genes with optimized RBSs. There are currently no in silico models for the selection of the most efficient RBSs for Synechocystis so we had to find one by trial and error and with help from literature. For our experiments we chose three different RBS sequences that had been found promising. By choosing an inducible promoter, the transcription of the genes and thus the bioremediation process are controllable.
The goal of our engineering is to give information and tools regarding mercury metabolism of Synechocystis and its use as a motor for converting methylmercury into elemental mercury. In addition, the work we did on the Golden Gate based assembly system gives valuable information about its function. This approach creates the possibility of efficient merA and merB gene expression in Synechocystis but in addition lays groundwork for research regarding mercury metabolism.
More information can be found on our Engineering, Design and Parts pages.
Synechocystis is an important photoautotrophic chassis for testing and learning, and
will be an important one in the future as well. The special attributes of Synechocystis make it a great platform for new renewable solutions, and work as a
stepping stone for widening the usage of cyanobacteria. The research done on Synechocystis can help expand the host range as the tools developed for it could be used
for biotechnological applications that need other cyano strains with more suitable qualities.
Synechocystis sp. PCC 6803 strain has been used in previous iGEM projects as well. We collaborated with the iGEM team of Edinburgh about using Synechocystis. We discussed the potential of our photoautotrophic chassis and in addition for example the time restrictions that the slower growth pace of the autotrophic Synechocystis has compared to the heterotrophic E. Coli. With good design Synechocystis is a very potential organism for future light-driven projects, also in the scope of iGEM.
You can read more about our collaboration with Edinburgh team on our Collaborations page.
New biotechnologies using photoautotrophic microbes are and will be developed for diverse applications. For instance, with our MercuLess truck, we can take part in ensuring clean habitats, ecological balances and the safe use of fish by remediating the mercury contaminated bodies of water with Synechocystis whilst using easily available energy sources, light and carbon dioxide. The development from the current situation into a future with different photoautotrophic organism based applications requires wider research, development of infrastructure, time and investments. Still our research, as each one in this field, takes us toward a light-driven future.
You can read more about our MercuLess truck on our Implementation page.
Singh, D. K., Lingaswamy, B., Koduru, T. N., Nagu, P. P., & Jogadhenu, P. S. S. (2019). A putative merR family transcription factor Slr0701 regulates mercury inducible expression of MerA in the cyanobacterium Synechocystis sp. PCC6803. MicrobiologyOpen, 8(9), e00838. https://doi.org/10.1002/mbo3.838https://doi.org/10.1002/mbo3.838