Team:Linkoping Sweden/Engineering
During our project, we came across multiple difficulties that forced us to rethink our plan and the way we do things. This is a natural part in any experimental research and it can be viewed as a part of the engineering cycle.
Figure 1. An illustration of the engineering cycle.
Synechocystis sp. PCC 6803 Growth
At the beginning of our project, we decided to use the halophilic and photosynthetic cyanobacteria Synechocystis sp. PCC 6803. Our reasoning was that it is a relatively well-established organism for synthetic biology. Synechocystis sp. PCC 6803 was also used by the SJTU BioX-Shanghai 2015 team for their project, which originally inspired us to choose desalination as the main goal of our project. During our growth curve experiments, we learned that Synechocystis sp. PCC 6803 was unable to grow for a long period of time in the non autoclaved sea- and brackish water. This led us to look at the samples under a microscope to try to figure out why our samples died. To our surprise, we found other phototrophic organisms thriving in our samples (Figure 2). We concluded that some other phototrophs or combinations of phototrophs could be more suitable for desalination. You can read more about the theorized phototrophs on our results page.
Figure 2. A. Comparison of Synechocystis sp. PCC 6803 grown in BG-11 with an open atmosphere (left), BG-11 with a closed atmosphere (middle), and non autoclaved brackish water from Gotland (right). B. Fluorescence microscope picture of the non autoclaved brackish water sample from Gotland. Chlorophyll in the bacteria was excited with a wavelength of 480 nm. The circled organisms are identified as 1) Unknown, 2) Aphanizomenon.
From the same water samples, we were also able to spot some potential predators that were killing the Synechocystis cells. From literature, we were able to possibly identify the predators as some kind of amoeba. We also found possible ways to genetically modify Synechocystis to make it more resistant against the amoeba [1]. This would be an alternative way to improve our project and overcome the problem with cultivating bacteria in non autoclaved water.
We also had to adapt our cultivation setup for the Synechocystis sp. PCC 6803 during our experiments. Our original idea was to cultivate the bacteria as liquid cultures in Erlenmeyer flasks sealed with aluminium foil. We soon discovered that our liquid cultures were not growing as dense as we hoped for. After some research and input from experts working with cyanobacteria, such as Dr. Julie Zedler and Dr. Vamsi Moparthi, we decided to switch the aluminium foil to cotton plugs. This allows proper gas exchange into the flasks, providing the bacteria with more CO2 which is needed for photosynthesis.
Toxicity
When trying to amplify the halorhodopsin construct in Escherichia coli (strain DH5ɑ), we noticed that the colonies grew slower than expected and remained extremely small. We made the same observations when halorhodopsin was transformed into Vibrio natriegens (strain Vmax), leading us to believe that halorhodopsin is toxic for both bacteria. When analyzing a number of our halorhodopsin constructs through sequencing, the results showed that the sequence encoding halorhodopsin was not conserved in our plasmids. The last part of the gene had been mutated in various positions in all products, causing a significantly shorter halorhodopsin to be expressed. Based on these results, it is plausible that halorhodopsin is toxic to its host organism and that the sequence becomes mutated in order to preserve the survival of the host organism. The possibly toxic effects of halorhodopsin is something that requires further research to be confirmed, but also something that should be taken into consideration in future research done using this ion pump.
Constructs
Another difficulty came with ordering our constructs. The plan was to have the constructs we designed synthesized as a part of a plasmid but unfortunately, the synthesis company failed to produce most of our constructs. This led us to order linear constructs with less complexity, meaning we removed the termination sequences, and ligated these fragments into plasmids ourselves. As a result of cloning our constructs into plasmid ourselves, we needed to have a working system to select the colonies with the correct insert. Our solution was to use pUC19, a plasmid commonly used for blue-white screening, but that we had not previously planned on using. The use of blue-white screening aided us in our laboratory work a lot. Furthermore, many failed ligation attempts led us to assume a new approach. In order to make our cloning more efficient, we decided to treat our plasmids with calf intestinal alkaline phosphatase (CIP). CIP catalyzes the dephosphorylation of 5’ and 3’ ends of DNA phosphomonoesters [2]. The dephosphorylation prevents the self-ligation of the linearized plasmid. This eliminates the possibility of getting colonies with a self-ligated plasmid with no intended insert.
[1] Simkovsky R, Daniels E, Tang K, Huynh S, Golden S, Brahamsha B. Impairment of O-antigen production confers resistance to grazing in a model amoeba-cyanobacterium predator-prey system. Proceedings of the National Academy of Sciences. 2012;109(41):16678-16683.
[2] New England BioLabs. Quick CIP [Internet]. New England BioLabs inc. [cited 17th October 2021]. Available from: https://international.neb.com/products/m0525-quick-cip#Product%20Information.