Difference between revisions of "Team:Exeter/FMR"

Fluidised Media Reactor (FMR)

A As a proof of concept experiment, we tested the ability for type I pili, in an MG1655 E.coli strain, to bind to mannose in a fluidised media reactor (FMR), as this could be investigated at the same time as the wet lab work was being developed. The bacteria formed biofilms on polypropylene torus scaffold structures within the FMR, with water containing mannose being passed through the FMR and the concentration removed being measured. This would show whether it was feasible to use this set up in a real life scenario.

Results

In order to optimise the parameters of the FMR, we used design of experiment (DOE). DOE is a systematic method to determine the relationship between factors affecting a process and the output of the process. In this case we will use this DOE to find the set of factors that removes the most mannose. After showing this worked our aim was to then test our own constructs in the same manor.

Media

The idea of using an FMR was introduced to us after a visit to Taunton Aquarium Centre. We visited here to obtain expertise on water filtration on small scales, where they have a variety of bioremediation techniques for removing harmful substances, such as nitrates and phosphates, from water to keep fish tanks healthy. They recommended a fluidised media reactor, which is a cylindrical vessel, containing media for bacterial growth, with the ability to be easily scaled up to suit a range of scenarios. In this case they used NP-Bacto-Pellets as media, which hosts nitrate metabolising bacteria that are already present in the water. They recommended to us to use silica sand in our experiments as the sand still function in low pH water, such as the ones found in the Wheal Maid site where one of the lagoons was at pH 2.80±0.03.

Following these discussions we investigated the use of silica sand in a fluidised media reactor. We sieved the sand so that we had three different size categories of sand: 150-180μm, 180-250μm and 250-425μm in diameter. This was in order to investigate how the size of sand grain affected the reduction in mannose concentration in the DOE. When running the FMR with sand as media we discovered that when water was pumped through, a large amount of sand ran out of the FMR. This is not sustainable as the mass of sand in the reactor steadily decreased. We decided that we must consider implementing some of the following factors to successfully fluidise the media:

26^th July

An electrophoresis was carried out using the amplified DNA from the previous day. As shown, the MG1655 reference is positive for all threefim genes that were amplified, while Top10 is negative for all three. This confirmed that Top10 was a more suitable candidate with regards to its genome.

28^th July

Overnight cultures of MG1655 and Top10 were removed from the incubator and were taken to the Bioimaging department. The samples were prepared with a negative stain and were placed on a small, circular copper grid before being inserted into the transmission electron microscope. Severalexposures were taken, as shown and these are demonstrated both that MG1655 does indeed produce pilus structures and that pili are absent from the surface of the Top10 strain. This, combined with the electrophoresis evidence, confirmed that the Top10 strain is the suitable chassis for our project.

August 2017

2^nd August

The fim operon + pAra MoClo products were cut using restriction endonucleases before being run down an agarose gel. The result gave confirmation that the fim operon had been transformed wholly into dh5ɑ in pSB1A3.

31^st August

The first attempt at the yeast agglutination protocol was made. Overnight cultures of Saccharomyces cerevisiae were incubated at 30°C with varying volumes of Top10 WT and MG1655WT. They were initially kept in the shaking incubator and were then transferred to a water bath at the same temp overnight. The MG1655 samples demonstrated agglutination by showing a pellet, while the Top10 samples did not.

September

1^st September

The first attempt at the yeast agglutination protocol was made. Overnight cultures of Saccharomyces cerevisiae were incubated at 30°C with varying volumes of Top10 WT and MG1655WT. They were initially kept in the shaking incubator and were then transferred to a water bath at the same temp overnight. The MG1655 samples demonstrated agglutination by showing a pellet, while the Top10 samples did not.

6^th September

The solutions of induced bacterial cultures and metal ions (of varying concentrations) were prepared and pipetted into Snakeskin dialysis tubing. The tubing was clamped and suspended in a beaker of MOPS buffer solution. The beakers were placed in a shaking incubator for 1 hour at 20°C and 100rpm. After this period, the MOPS solution was removed and sampled for later ICPOES readings. The MOPS solution was replaced and the previous step was repeated twice.
The samples were run through the TECAN for fluorescence as done on the 1st.

References