Proof of Concept
Our team has developed the spacer system to facilitate the insertion of multiple large genes into a construct. The spacer system acts as a buffer between the genes and consists of the forward and the back spacer. The system design also implied having a ribosome binding site and unique enzyme cutters for easier detection of the genes presence during restriction digestion. We implemented this system with all the genes of interest, resulting in fewer primers needed, and reduced PCR requirements prior to Gibson assembly. The presence of multiple RBS, embedded in the spacer system will allow for simultaneous translations. These are crucial for developing different combinations of the genes.
Primers were tested in different combinations, allowing for different PCR products with varying overhangs. Gibson assemblies were run based on this and later plated on agar plates. Successful application of the spacer system was achieved for two genes. After plate growth was detectable, re-plating, mini-preps and restriction digests were conducted. These restriction digests targeted the sequences included in the spacers. Bands were compared to the supposed gene length with the spacer attached. This proved that the spacer system worked as desired. For further proof, the mini-prep products were sent for sequencing, which once more confirmed the effectiveness of our spacer system.
To test our transformed bacteria in future experiments, we had to design a simulation in vitro of a cows rumen. This simulation had to have the following attributes:
Anaerobic conditions – Digestive tracks are generally anaerobic, therefore such an environment was created using glassware, silica gel, airtight parafilm and an oxygen gas meter.
Addition of saliva buffer and removal of liquids – Cows continuously produce saliva and swallow it into their digestive tract, where it leads to the rumen together with their feed. Within the rumen, fermentation processes allow for the production of volatile fatty acids, which can be used as an energy source by the cow. Yet fluid also is removed. Therefore, the standard protocol, or SP for short, was developed. Hourly, saliva buffer was added and ruminal fluid was removed. This was conducted with minimal oxygen added to the system. Afterwards, the system was flushed with nitrogen to remove any oxygen that may have been inserted during the removal of liquids. Furthermore, feed in the form of concentrate bags containing grass was added every 24 hours.
In one of the two simulations, pure bromoform (96%) was added to simulate the addition of our transformed bacteria.
After measuring over the timespan of two days, it was determined that the bromoform-treated simulation had indeed reduced gas production. This was assessed using inverted glass flask in a water filled bucket.
With this successful experiment, we have obtained proof that this setup works and is a viable testing device for further proof of concept by adding our bacteria instead of bromoform to measure their methanogenesis inhibiting ability. Furthermore, this is additional proof of bromoforms ability to reduce gas productions in the rumen. Additionally, in future experiments, this setup can also be used to asses the function of our biosafety aspects by testing whether the plasmid has spread to different microbial species, and by testing the survival rate of any of our bacteria outside of said simulation in conditions that should trigger the kill switch.
Yet further testing needs to be conducted to establish the specific methane content differences between the two simulations.
More details about our experiment using ruminal fluid in these simulations can be found under Model.
Furthermore, with the help of our tutorial on how to set up this simulation, we have added a valuable contribution for future teams that wish to conduct similar experiments in anaerobic conditions. These tutorial videos can be found under the Hardware award.