On the 1st and 2nd of August, we presented a stall at Glasgow Science Centre as part of their ‘Meet the Experts’ event. This was our first chance to reach out and engage with the public to get real feedback about the things we had planned, which was overwhelmingly positive!
We used this opportunity to find out what our target market thought of Furri-Lux – to help with this, we had a large colouring-in picture of Furri-Lux’s logo for younger children, as well as blank paper for older kids to design their own ‘friendly monster’, all of which we displayed on a poster board next to our table.
These drawings later became inspiration for Furri-Lux’s outer design, as we were particularly interested in which features children associated with ‘friendly’ and ‘scary’ monsters.
For their parents, we had flyers with information about iGEM, the competition, past projects as well as our project to take away and read at their leisure, along with an interactive ‘circuit quiz’ on how the bioluminescence system worked inside the cells. The right answer had to be placed onto the board for it to conduct, and light up the LED!
On the whole, many parents appeared very receptive to the idea of a children’s product incorporating bacteria, which was greatly encouraging. A common theme seemed to be that many parents believed their children were already exposed to a lot of much more harmful bacteria in every day life, so more education for younger kids was definitely needed. **Should I keep this next part?** However, their concern was apparent when we tried to talk to the children about ‘monsters under the bed’ – clearly this was a fear that parents were not immediately willing to validate, and they had put a lot of effort into trying to convince their children that these ‘monsters’ didn’t exist. Therefore, buying a product that offered help with this problem seemed contradictory and counter-productive. We at iGEM Glasgow, on the other hand, feel that validating the children’s’ fears is more important to their healthy emotional development, as well as strengthening the parent-child bond as they work through it together.
Parents also expressed concerns that curious children would try to open the container themselves – which led us to design the removable handle for the bacteria’s release. To read more about this, see our Product Design page.
Hyndland School Visit
We had the chance to invite some senior pupils from the nearby Hyndland Secondary School to our lab, so that they could learn more about the iGEM competition and the other opportunities available to them at university.
Afterwards, we let them practice their aseptic technique by drawing their own pictures on agar plates with our glowing bacteria – and they came up a treat!
Most of the pupils were studying a course on Lab Skills, so we showed them around the lab we were working in and discussed the operation of some of the equipment they may not have had substantial access to in school – such as the autoclave and flow hood.
The remaining pupils were from their Advanced Biology course, who found the more in depth presentation of our bioluminescence system to be more interesting and insightful.
Glowing Plants Controversy
Originally, the system containing UirS, UirR, and PlsiR accounts for a negative phototactic response to unidirectional UV-A light. The proposed mechanism puts UirS, a transmembrane protein of the CBCR family, as the molecule that perceives UV light. It is suggested that through a physical interaction between UirS and UirR and possibly a phosphotransfer from UirS to UirR, UirR is released from the transmembrane protein. The released UirR can now bind to DNA and UirR, which is similar to other activators of stress responses, was found to be a transcriptional activator of lsiR after binding to its promoter PlsiR .
We suggest a system where UV-light triggers the expression of a repressor that acts on the production of bioluminescence genes to alleviate the burden they may cause on the cells’ metabolism if constantly expressed. During the night, the bioluminescence genes are expressed, and produce a green-blue light. As it becomes day, UV-A causes the release and activation of UirR. UirR binds to PlsiR to turn expression of the repressor PhlF. PhlF binds to PPhlF to turn off expression of LuxCDABE, so there is no bioluminescence. As it becomes night again, UirR is no longer bound to PlsiR so expression of PhlF is turned off. As PPhlF is no longer repressed, expression of LuxCDABE is turned and bioluminescence is produced again.
We have confirmed through the use of a laser scanner that PlsiR is not active when UirS and UirR are absent. PlsiR was ligated to GFP with two ribosome binding sites of different strength and no fluorescence was observed (the parts we used for this experiment were K1725401 and K1725402) (Chart 1). Moreover, cells that possess UirR but lack UirS also did not show levels of fluorescence above the expected for E. coli. Therefore, UirR is not sufficient to drive the activation of PlsiR.
Chart 1. Relative Fluorescence (Compared to Last Taken Measurement of Constitutively Expressed GFP Control) over Absorbance in DH5α cells. DH5α cells containing the PlsiR promoter with GFP fluoresce no more than the original laboratory strain or cells that have GFP without a promoter.
Survey Analysis
Overview
We conducted a survey of 60 random members of the public to attempt to gauge how willing they would be to give our night-light to a child, and to ascertain their overall view of genetically modified organisms. A full list of the questions asked can be seen here.
In particular, we were looking at:
The demand for a children’s product in this genre
The respondents’ level of science education
How strongly people agreed or disagreed with 7 points:
I would allow my child to have an educational toy that needed to be fed and looked after once a day with my supervision.
I would allow my child to have an educational toy that incorporates E.coli bacteria.
I would allow my child to have an educational toy that incorporates genetically modified bacteria.
I would allow my child to have an educational toy that incorporates bacteria.
All strains of E.coli are dangerous.
E.coli regularly exists in my body.
If these genetically modified bacteria were accidentally swallowed by someone, they would become dangerously ill.
For the 7 opinion points, we first assessed how they felt about them as it stood in that moment with their current level of knowledge on the subject, then allowed them to read more information about the bacteria we were using in our project:
What is 'synthetic biology'?
Synthetic biology is a branch of biology that combines the powers of biotechnology, evolutionary biology, molecular biology, systems biology, biophysics, computer engineering, and genetic engineering (to name but a few) in order to construct 'biological devices' or 'machines' that are useful for solving problems - anything from something as trivial as chewing gum on the streets to solving climate change.
What are e.coli?
E.coli bacteria are commonly found in the lower intestine of warm-blooded organisms (in other words, you and I!) where they make up the normal flora of the gut, produce vitamin K2, and prevent dangerous bacteria from growing. Most strains of e.coli are completely harmless, however there are one or two types that cause serious food poisoning - because of this, many people understandably assume that just because it's called 'e.coli', it must be very harmful, however this isn't the case!
In our labs, classes and research, we use e.coli as they grow quickly and are easy to look after. The bacteria we use are a 'lab strain' which means we know exactly what their genotype is (the full set of genes present in the organism), and any and all dangerous genes or features that could cause it to infect humans and cause illness have been removed.
What are you doing to it?
Over the summer we have been working on taking bioluminescence genes out of a bacterium called Aliivibrio fischeri and genes from a cyanobacterium called Synechocystis that can detect UV-A light from the sun, and placing them in our e.coli cells.
When combined with some other standard genetic parts, the result is e.coli bacteria that glow when it's dark!
What are you using it for?
Our project involves designing a toy nightlight for children based around the idea of a "friendly monster", i.e. one that scares away the bad monsters under the bed! This would involve a stuffed monster toy, inside which would be a clear plastic container that would hold our bacteria. Every morning the child, with parental supervision, would turn a tap at the base of the toy, and the nutrient solution containing the bacteria would drop out of the bottom into the toilet to be flushed away. The handle of the tap connects via a key slot so that curious children can't open it and spill everything everywhere on their own. The child can then "feed" their monster friend by pouring fresh nutrient solution into their mouth, which passes through a tube through a one-way valve down into the main part of the vessel, so that if it were dropped or accidentally turned upside down the bacteria and solution would stay inside. There would be enough bacterial residue stuck to the inside of the container to allow them to repopulate during the day while the child is at school or nursery. Once they no longer detect the UV-A from the sun, the bioluminescence system becomes activated and they will begin to glow. At night the child would give the monster a gentle shake, which would allow oxygen to distribute more evenly among the bacteria encouraging brighter, more even bioluminescence.
Kids are mischievous and curious, what if they tried to eat it?
The bacteria are harmless, as is the nutrient solution they'd be in. The environment in the stomach would kill them immediately. However, it wouldn't taste very nice at all, which may cause them to bring it back up. Ultimately the child would come to no lasting harm and would not require professional medical treatment, just a big glass of water to get rid of the taste.
The same questions were then asked again, in order to see if providing more information about the details of our project would alter the public’s view on these points.
Initial aims
Initially we wanted to explore what happened over a relatively small period of exposure, as our assumption was that we would see substantial reduction in a fixed number of E.coli, even over a relatively small time period. We also wanted to see how the different strains (DH5α ,TOP10) being used would respond compared to each other.
Since both DH5α and TOP10 are recA negative, and are therefore incapable of certain major kinds of DNA repair, we predicted that these strains would be more acutely affected than DS941 and MG6155.
Method
1ml 10x serial dilutions were made up to 10-6 from a 5ml overnight of each strain. 10µl spots of each dilution were then spotted onto LB agar plates.
These plates were then exposed to 50µmoles/m2/s of UVA at room temperature in illumination cabinets. Time points were then taken by removing plates from the illumination cabinet.
After illumination, plates were incubated at 37°C, under the assumption that every single viable cell will form a colony. The length of incubation is irrelevant, provided that every cell is given enough time to form a visible colony. This also forms the basis of our counting system, where a colony is assumed to have come from a single cell. We also make the assumption that cell division at the temperature and time we were running the experiment was negligible/nonexistent.
After incubation, the colonies on each spot/dilution were counted. The number of colonies from the lowest visible dilution (some dilutions formed a lawn of growth) were then multiplied by the dilution factor to approximate how many cells would be in 10µl of undiluted culture.
Results
Figure 1 mean estimated cell count per 10ul of a 5ml overnight in lb broth over time of exposure to 50 µmoles/m2/s of UVA time points taken at 010 20 30 60 120 . time points connected by straight line
We seemed to be seeing a decrease by 30 mins and between 60 and 120 at least in the recA positive strains the decrease between 0-30 is much steeper in the reca negative strains (fig1).
Since we had no time points between 60 -120 we decided to take time points at 80 100 120 to better visualise the change (fig 2).
Fig 2 mean estimated cell count per 10ul of a 5ml overnight in lb broth over time of exposure to 50 µmoles/m2/s to UVA time points at 0 80 100 120 time points connected by straight line
As noted above figure 2 show this second decline seems to be a feature of MG6115 and DS941, may be du e to their recA status
Figure 3 estimated cell count per 10ul of a 5ml overnight in lb broth over time of exposure to 60 µmoles/m2/s to UVA composite figure including mean from previous 2 graphs
It’s reported that E.coli suffer lethal effects at around 1000kw with illumination at 366nminin continuous culture (Berney et al 2006).this corresponds with around 16 hour with the fluence and wavelength we were using. We decide to illuminate bacteria for 14hours as we felt that if least some bacteria could withstand 14 hours of activating radiation then the idea as using UVA as the input into the toy was at least theoretically feasible
Plates at 14 hours showed growth
We have confirmed through the use of a laser scanner that PlsiR is not active when UirS and UirR are absent. PlsiR was ligated to GFP with two ribosome binding sites of different strength and no fluorescence was observed (the parts we used for this experiment were K1725401 and K1725402) (Chart 1). Moreover, cells that possess UirR but lack UirS also did not show levels of fluorescence above the expected for E. coli. Therefore, UirR is not sufficient to drive the activation of PlsiR.
