Team:Exeter/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.

July 19th
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:

  • Larger media – as this requires larger flow rates to fluidise.
  • Lower flow rates – this would reduce the sand that flows out the FMR.
  • Finer sponges – to prevent sand escaping the FMR.

The most convenient of these options was to use a finer sponge that would not be able to pass sand through. For this we used cellulose sponges, placing a thin disk at the bottom and at the top of the FMR. Due to the properties of the cellulose sponge, it created a resistance to the flow rate, which meant flow rate reduced when using these sponges so we had attempted to address two of the three factors we had intended to. When pumping water through the FMR with this new set up, we saw an improvement in the sands ability to fluidise, but the large resistance to flow caused by the sponges often led to reliability issues with the pump, sometimes leading to no water outflow. We observed the fluidising of the sand in some regions, but could not make the fluidising uniform due to the cellulose sponge not being uniform in shape, causing flow to arise mainly from one side.

July 26th

While investigating the behaviour of the silica sand in the FMR, we also looked into the ability of MG1655 to form biofilms and grow on the surface of the sand. We did this by growing MG1655 overnight on silica sand overnight in an LB medium. We then prepared the samples for SEM, imaged them and compared the growth to that we observed under the same conditions on NP-Bacto-Pellets, as well as negative controls for both. Initial imaging of these were unsuccessful as the overnights had been left for five days before they were eventually imaged, causing the bacteria to die from lack of nutrients, as well all the samples getting contaminated. This imaging process was then repeated and produced a density of 0.0088±0.0014μm-2 for sand and 0.2±0.4μm-2 for the NP-Bacto-Pellets. The large range in value for the NP-Bacto-Pellets was due to one image having a large number of MG1655, while the rest all had none or very few. Initially it appears that less biofilms are able to be formed by MG1655 on the sand and the NP-Bacto-Pellets, however it is not certain due to the large uncertainty of the latter.

August 2017

2nd 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.

August 2nd
31st 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

1st 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.

6th 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