The third component of our system is a biosecurity mechanism to ensure the containment of the genetically modified bacteria, a crucial component for any synthetic biology project. For this final component we have explored different methods of biosecurity and conducted a number of experimental tests to determine its suitability within our innovation.
Testing the effectiveness of UV as a method of Biosecurity
The aim of this set of experiments was to determine the effectiveness of using Ultra Violet radiation as a bactericide to prevent the release of our GM E.coli into the environment in addition to preventing other bacteria from entering the filtration system. The idea of incorporating UV in our bio-security scheme came from a trip to a South West Water plant. Click here to find out more . At that particular plant bacteria were used to digest organic waste. Before water could exit the works it flowed under UV light to remove bacteria that were remaining. However the bacteria used by SWW is not genetically modifies and is naturally occurring. As stated above we however are proposing to use GM bacteria and therefore we must adhere to UK laws and standards. Therefore it was important to test the effectiveness of UV light to see if it could be suitable to use in our filtration system.Click here to find the protocol we devised!
As part of our research into finding the most appropriate and effective biosecurity mechanisms to include within our filtration system, see link for more information, we decided to firstly test how the population of E. coli changes with exposure to UV. The motivation behind testing this particular mechanism was because it was mentioned in a interview with South West Water as part of our stakeholder engagement that this methods was employed at one of their works which use bio-remediation in its producers, click here to find more information about his interview.
The results of the experiment are as follows.
|Time Exposed(minutes)||Average percentage of the control surviving (%)||Average percentage of the control killed (%)|
Both graphs clearly demonstrate that 10 minute exposure to the UV light caused the greatest cell death, suggesting that greater the exposure time the greater the number of cells killed, as we would expect. Most significantly for our filtration though, this experiment has proven that the use of UV radiation is not a suitable bactericide. This is because in order to achieve an escape frequency anywhere close to the current legal requirement (which is of the order 10-8) the water would have to be irradiated for a period much longer than 10 minutes which is not practical for our intended use. Therefore we need to explore possible alternative to UV.
Whilst plate counting it was noticed that some cells on the plates were larger than others, (see image image below). At first, as this had not been observed before, we assumed it was because we had left the plates in the incubator for too long which had allowed for them to grow to such a size. However after reducing the time the plates were incubated for and still seeing the same results, we consulted our PIs. It was suggested that these anomalies could be a result of mutations where the UV was not powerful enough to destroy the cell but had managed to damage the cell's DNA. This idea is supported by the fact that the observed larger cells were most common on plates that had been irradiated for 5 and 10 minutes. The occurrence of these mutated cells strengthens our claim that the use of UV is not suitable. This is because it has the potential to cause further mutations to the E. coli's DNA which could bring about unforeseen impacts to the surround ecology.
In a collaboration with the University of Cardiff iGEM team further experiments were repeated. Specifically Cardiff were able to measure the number of E. coli that had survived at additional exposure times of 1 minutes, 20 minutes and 30 minutes in addition to repeating the exposure time we tested.
Cardiff repeated the experiment for a 1 in 100,000 dilution and went on to further conduct it using both a 1 in 10,000,000 and in 1 in 100,000,000. However the results from the further two dilutions showed these dilutions did not yield populations of colonies in which we could apply statistical methods of analyse to produce meaningful results. All data that Cardiff contained for us is contained in the table below.
The work carried out by Cardiff supports our claim that the use of UV as a bactericide would not be appropriate within our filtration system as exposure time would be too great when the amount of water we wish to process.
In collaboration with Newcastle the investigation into the effective of UV as bactericide however due to restriction on the device used to generate their UV light the maximum amount of time that they could irradiate the sample for was 10 minutes. This being said the results they obtained for 1 minute, 2.5 minutes , 5 minutes and 10 minutes of exposure were not as expected. Below is a description of our engagement with Newcastle using the AREA framework detailed on our Silver Human Practices page.
Our communications with Newcastle were mainly over email in addition to one skype call at the start of the collaboration where we discussed what we could do for each other.
We assumed that the results would be similar as the protocol was detailed about how to conduct the experiment and what devices should be used.
Although the data differs from us it demonstrates the difficulty in scientific communication. It has further motivated us to collaborate with another team to see what their results would be.
Despite not getting the results we had expected this informed our further collaborations, specifically with Cardiff, to ensure the protocol was more explicit and to continuously make ourselves available for questions.
As a result of a meeting with one of our key stakeholders from the Plymouth Marine Laboratory (PML) we were introduced to the use of a 'Vortex Bio-reactor'
in conjunction with an anti-microbial agent as an alternative form of bio-security. The anti-microbial agent used by the PML were copper alginate beads. The investigation they had
conducted was to compare between the use of UV and copper alginate beads within a Vortex Bio-reactor as an effective pathogen destruction technology. It was
found that the use of copper alginate beads was superior to UV and significantly so when applied to organisms resistant to UV-C.
This is seen in graph below where the UV resistant organisms, D. radiodurans, shows a significant decrease in survival rate between the two techniques.
However concern has been expressed over the use of copper as a bactericide as the copper must adhere to the cell surface in order to kill the cell. This of course could prove problematic in that our project, which aims to bind metals ions to the protein on the ends of pili, would reduce accessibility to cell surface. In addition to this the authors of the paper have mentioned that more research surrounding the longevity and robustness of the beads is needed to quantify the risk of the beads breakdown and releasing copper back into the water.
Another possible alternative to UV that we are considering is the use ozone. However we choose not to explore this method due to concerns over it contaminating/reacting when used in high concentrations.
Simon F. Thomas et al, 2014. A Comparison between Ultraviolet Disinfection and Copper Alginate Beads within a Vortex Bioreactor for the Deactivation of Bacteria in Simulated Waste Streams with High Levels of Colour, Humic Acid and Suspended Solids. PLOS 0115688.g006.