|
|
| (40 intermediate revisions by 4 users not shown) |
| Line 1: |
Line 1: |
| − | {{Exeter/header}}
| |
| − | {{Exeter/navbar}}
| |
| | | | |
| − | <html>
| |
| − |
| |
| − |
| |
| − | <!-- Row & container for main content of the page -->
| |
| − | <div class="container-fluid">
| |
| − | <div class="row">
| |
| − | <div class="col-2">
| |
| − | <a href="https://2017.igem.org/Team:Exeter"><img id="logo-top-left" width="100%;" src="https://static.igem.org/mediawiki/2017/2/29/T--Exeter--startpage_logo.png"></a>
| |
| − | <nav id="navbar-page" class="navbar"> <!-- classes removed from template: navbar-light bg-light-->
| |
| − | <!-- <a class="navbar-brand" href="#navbar-top">Back to top</a> -->
| |
| − | <nav class="nav nav-pills flex-column">
| |
| − | <a class="nav-link" href="#Results">Results</a>
| |
| − | <a class="nav-link" href="#Media">Media</a>
| |
| − | <a class="nav-link" href="#HPLC">HPLC</a>
| |
| − | <a class="nav-link" href="#DOE">Design of Experiment</a>
| |
| − | <a class="nav-link" href="#navbar-top">back to top</a>
| |
| − | </nav>
| |
| − | </nav>
| |
| − | </div>
| |
| − | <div class="col-10" id="pageContent">
| |
| − | <div>
| |
| − | <h3 id="pageHeader">Fluidised Media Reactor (FMR)</h3>
| |
| − | <p>The second component, of our applied design, is the <b>fluidised media reactor (FMR)</b>. The fluidised media reactor is a piece of cylindrical
| |
| − | housing apparatus designed to contain our genetically modified bacteria. The water, now free of large particulates, is fed into the top of the
| |
| − | FMR. It travels down through the central pipe, and slowly rises back through the outer pipe, in which the bacteria are contained. The <i>E. coli</i>
| |
| − | are grown on polypropylene scaffold torus structures, using a bio surfactant which stresses the bacteria and promotes the development of a biofilm. Here, we have optimised
| |
| − | the flow rate to ensure that the bacteria have a high probability of binding to the targeted metal ions. Additionally, we have created a mathematical model to inform the scalability of this aspect of the filtration system as it is unique to our project. <a href="https://2017.igem.org/Team:Exeter/Model">Click here to find out more about our model.</a> </p>
| |
| − | <p>As a <b>proof of concept experiment</b>, we tested the ability for type I pili, in an MG1655 <i>E. coli</i> 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 <b>polypropylene torus scaffold structures</b> within the FMR, with water containing mannose being passed through the FMR and the concentration removed being measured using <b>High-Performance Liquid Chromatography (HPLC)</b>. This would show whether it is a feasible choice of reactor for our purpose. </p>
| |
| − |
| |
| − | <img class="rounded mx-auto d-block w-50" src="https://static.igem.org/mediawiki/2017/2/20/T--Exeter--FMR.jpg" alt"Results">
| |
| − |
| |
| − | <h4 id="Results">Results</h4>
| |
| − | <p>In order to <b>optimise</b> the parameters of the FMR, we used <b>Design of Experiment (DOE)</b>. 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 <b>test</b> our own constructs in the same manor.</p>
| |
| − | <p>Ideally, we would have conducted this research using the constructs produced by the Harvard team. However, we could not be provided with the JW4276 <i>E. coli</i> strain, used by their team, until much later in our project and we could not express their construct in the Top10 <i>E. coli</i> strain we used until late in the project. </p>
| |
| − |
| |
| − | <h4 id="Media">Media</h4>
| |
| − | <p>The idea of using an FMR was introduced to us after a visit to Taunton Aquarium Centre <a href="https://2017.igem.org/Team:Exeter/HP/Gold_Integrated#Taunton_low_level">click here to find out more</a>. 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 <b>Wheal Maid</b> site where one of the lagoons was at pH 2.80±0.03. </p>
| |
| − | <p>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: </p>
| |
| − |
| |
| − |
| |
| − | <ul style="list-style-type:circle">
| |
| − | <li>Larger media – as this requires larger flow rates to fluidise.</li>
| |
| − | <li>Lower flow rates – this would reduce the sand that flows out the FMR.</li>
| |
| − | <li>Finer sponges – to prevent sand escaping the FMR.</li>
| |
| − | </ul>
| |
| − |
| |
| − | <p>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. </p>
| |
| − |
| |
| − | <p>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.</p>
| |
| − | <p>Our struggles with silica sand led us to visit the Plymouth Marine Laboratory (PML) <a href="https://2017.igem.org/Team:Exeter/HP/Gold_Integrated#PML_low_level">click here to find out more</a>, who have expertise mainly in the marine environment, but we had arranged to speak specifically with a research group who have experience with cultivating biofilms and bioremediation in the past. We were recommended to grow the bacteria on polypropylene scaffold structures, using a chlorhexidine gluconate surfactant, to stress the bacteria to induce biofilm production. This led us to move away from using particulate media as a growth surface. Armed with this information we loaded the fluidised media reactor with polypropylene scaffold structures that we modified into tori (see figure). When water was pumped through the FMR the issue of media escaping was no longer an issue at any of the flow rates the pump produced. This led us to make the decision that we would focus on these scaffold structures instead of silica sand, providing we could successfully grow biofilms on the structures.</p>
| |
| − | <p>In order to do this we investigated a range of factors that would affect growing conditions:</p>
| |
| − |
| |
| − | <ul style="list-style-type:circle">
| |
| − | <li>Length of time left in incubator.</li>
| |
| − | <li>Angular frequency of incubator.</li>
| |
| − | <li>Concentration of surfactant.</li>
| |
| − | </ul>
| |
| − | <p>Testing the first two factors required a method of measuring the biofilm growth on the tori. The simplest method for this is to have an observer view the tori after growth to gain a qualitative opinion on biofilm growth. Although this lacks scientific rigour, it was the only viable method to produce results quickly due to time restraints. These factors were deemed less important than the final factor, so we deemed a less rigorous method acceptable. We determined that incubating the tori for 22 hours was most practical for our experimentation, and gave suitable time for biofilms to be formed. Secondly we found that incubating the tori statically produced better biofilms.</p>
| |
| − | <p>For last of these factors, we tested a range of concentrations and measured the biofilm growth by two methods. Firstly, we imaged the tori using SEM before and after they had been used in the FMR. This meant the density of bacteria on the surface of the tori could be measured. Should there be a similar number density in these samples, it shows the bacteria remained adhered to the surface after fluid had been passed through it, so the biofilms had formed well. However, due to the large uncertainty previously seen from the density of bacteria on other mediums, the images alone may not come to a conclusion. The mannose concentration in the water was then measured to indirectly investigate the biofilm growth. The larger the concentration reduction, the more MG1655 are present on the sponge, so the more biofilm growth. Should both these results support the same conclusion we will find an optimal concentration of surfactant to use.</p>
| |
| − | <p>One of the SEM images can be seen in figure with the results of the number density of MG1655 at different surfactant concentrations before and after use in the FMR is shown in table 1. The data from here shows that the bacteria density is largest for the 0.1% surfactant concentration, and smallest at 5%. It also shows the reduction in bacteria density following being used in the FMR. All three of these reduction values have an uncertainty larger than the value itself suggesting that it is statistically possible that the density did not change significantly before and after use. This suggests the MG1655 remain adhered to the polypropylene surface. However due to the large uncertainty it is also possible that a significant proportion of the bacteria was removed from the surface following use. Thus further work must be conducted to investigate this further.</p>
| |
| − |
| |
| − | <div style = "width:60%;margin: 0 auto; float:centre;">
| |
| − | <figure>
| |
| − | <img class="rounded mx-auto d-block w-50" src="https://static.igem.org/mediawiki/2017/f/fb/T--Exeter--Plastic_SEM.jpg" style="width:500px;margin:0 auto;"/>
| |
| − | <figcaption style="text-align:left">Figure ? - Times 5000 magnification SEM image of MG1655 growing on polypropylene scaffold structures at 0.1% of chlorhexidine gluconate surfactant.</figcaption>
| |
| − | </figure>
| |
| − | </div>
| |
| − |
| |
| − | <p> The mannose concentration reduction was measure using high-performance liquid chromatography (HPLC), which is discussed in more detail in the next section. The result measuring the reduction at different surfactant concentrations can be seen in figure ??. The reduction was found by measuring the initial concentration of the mannose before running through the FMR, and measuring the final concentration of the mannose after 1L had passed through the reactor. Figure ?? conclusively shows that 0.1% surfactant produced the greatest reduction in mannose concentration so was the most effective at inducing biofilm growth on the polypropylene scaffolds out of the three surfactant concentrations investigated. It is likely that the higher concentrations of surfactant at 1% and 5% began to cause cell death. </p>
| |
| − |
| |
| − | <img class="rounded mx-auto d-block w-50" src="https://static.igem.org/mediawiki/2017/7/78/T--Exeter--MW_test.jpg" alt="Media">
| |
| − | <h6> Figure ? - Graph showing how the concentration of chlorhexidine gluconate surfactant, used to induce biofilm formation on the polypropylene scaffold structure, is related to the reduction in mannose concentration after 10g/L mannose solution was passed through the fluidised media reactor. </h6>
| |
| − |
| |
| − | <p>Although the data points on figure ?? suggest a linear fit, it cannot be assumed. We were led to believe there would be a peak in the curve showing an optimal concentration of surfactant as too low a concentration would not stress the bacteria to produce biofilms while too high a concentration would kill the organisms. This peak is likely to lie in the region of 0-1%, and a more detailed analysis of this region would determine this. However for the purpose of this experiment, we decided to use 0.1% surfactant for the DOE, due to time constraints.</p>
| |
| − |
| |
| − | <h4 id="HPLC">High-Performance Liquid Chromatography (HPLC)</h4>
| |
| − |
| |
| − | <p>In order for DOE to be successful, a reliable and accurate method is required to measure the concentration of mannose before and after the solution has passed. We decided to use high-performance liquid chromatography (HPLC) which measures the absorbance of a sample which can be matched to a concentration of that sample from a concentration-absorbance calibration curve. We did this initially for mannose to make a calibration curve and understand the concentration range we can use in the DOE. We would have liked the concentration range that we chose to investigate to have be strongly dependant on the results of the Wheal Maid field trip <a href="https://2017.igem.org/Team:Exeter/HP/Fieldtrips">click here to find out more</a>, which found that the majority of the metal ion concentrations, above the drinking water standard, in the water fell between 0.03-50mg/L. </p>
| |
| − |
| |
| − | <p>However due to delays created by the Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) machine which measured the metal ion concentrations in our water samples, from Greenpeace, breaking down. For this reason we considered the concentration of metal ions from other water sources that have suffered from acid mine drainage. The Philippines for instance, at the Lafayette mine, found creeks to have metal ion concentrations of cadmium to be 0.811mg/L, whereas the Philippines National Standards for drinking water have a maximum concentration for cadmium being 0.003, almost 300 times smaller than the safe levels. A key component of this experiment will be to see if the concentration of mannose can be reduced by a similar order of magnitude.</p>
| |
| − |
| |
| − | <p>We initially decided to produce a mannose serial dilution from 40g/L to 0.625g/L and run it through the HPLC with a water buffer. From this we will be able to establish the accuracy of the HPLC from the calibration curve it produces, and whether it is possible to run lower mannose concentrations through it. Three technical repeats were taken from this to give the results shown in figure… This shows one of the technical repeats. In this figure the peak produced by the mannose at each concentration had drifted from sample to sample which occurred in all the technical repeats. This indicates that there may have been a blockage that preventing the samples from being injected at the same time. The area of the peak at each concentration was averaged for the three technical repeats to produce the calibration curve shown in figure… The data points lie very close to the line of best fit, with a chai squared of…</p>
| |
| − | <img class="rounded mx-auto d-block w-50" src="https://static.igem.org/mediawiki/2017/c/c8/T--Exeter--ManTestPeaks.jpg" alt="Media">
| |
| − | <p>The drifting of the peaks is a concern for us to produce accurate results. For this reason some parts of the HPLC were replaced and we decided to use an acid buffer instead of a water buffer.</p>
| |
| − |
| |
| − | <h4 id="DOE">Design of Experiment (DOE)</h4>
| |
| − | <p>There were many parameters that were required to be investigated in order to optimise the extraction of metal ions from water. JMP (Design of Experiment software) was used to create this optimisation. Two responses were chosen to characterise the success of mannose extraction. These were the percentage of mannose removed and the change in concentration from the mannose solution prior to entry in the reactor. Three factors were investigated to optimise the responses: initial mannose concentration, flow rate through the reactor and the number of sponges in the reactor.</p>
| |
| − |
| |
| − | <p>By varying the level of interaction and also the number of runs of the experiment it was possible to create different designs. These designs were then scrutinised, in the Design Evaluation, to determine which would produce results with the most confidence. The key areas of Design Evaluation that were compared are: Power Analysis, Predicted Variance Profile, Predicted Variance Surface, Estimation Efficiency and the Colour Map on Correlations.</p>
| |
| − |
| |
| − | <p>We looked at several designs by looking at first, second, third and response surface methodology (RSM) interactions of the factors. Each of these were </p>
| |
| − |
| |
| − | <h4 id="references">References</h4>
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | </html>
| |
| − |
| |
| − | {{Exeter/footer}}
| |
