Team:Manchester/Demonstrate
DEMONSTRATION
"Is Cutiful viable?" You are right to ask yourself that question, and Manchester iGEM 2019 is proud to answer that: "yes, it is!". We have shown on the Colour and Fragrance pages the ability of DH5⍺ to successfully secrete chromophores and odorants, and here we want to prove to you that, not only does DH5⍺ attach to hair, it is also able to survive in saline and chlorinated environments - such as found in pools, or the sea - and resist a shampoo wash. After all this, as the Brits say: "Bob's your uncle"!
Our achievements …
In 6 bullet points
1.
Demonstrate: Bacteria attach to hair- we demonstrated that E. coli DH5⍺ and BL21(DE3) attach to hair
2.
Demonstrate: chromophore coats hair- we proved that our system works through coating natural hair samples with coloured bacteria transformed with our designed constructs. (Colour, Act II: Experiments)
3.
Demonstrate: Bacteria resist daily stress- we proved that bacteria do not die under realistic conditions: swimming in chlorinated or saline water. (Human Practices, Act V: Conversations with Experts)
4.
Demonstrate: Bacteria survive shampooing- we identified that E. coli does not significantly detach from hair in response to shampoo-washing and hence that the hair dye will not be removed by showering. (Human Practices, Act V: Conversations with Experts)
5.
Customisable-We highlight the customisable aspect of our product through its modular nature. The division of all aspects (colours, scent and repair) into different E. coli provides customers with a personalisable product.
6.
Variety of Hues-We have demonstrated that different ratios of bacteria can achieve a whole range of colours as requested by the public and our stakeholders. (Human Practices, Act II: The Community Festival; Act V: Conversations with Experts)
Prologue
Our initial approach in order to design a bacterial-based hair dye was to use a naturally attaching bacteria. Knowing that some E. coli ATCC 25922 had been already characterised as part of the hair microbiome, and were naturally attaching to hair (Kerk et al., 2018), we compared its whole genome to that of other E. coli strains which we had available in house using BLAST Ring Image Generator (BRIG).
It was decided that E. coli Nissle 1917, BL21(DE3) and DH5⍺ cells would be tested for hair adhesion, and we found that they have >98% genomic similarity to ATCC 25922. Whereas both BL21(DE3) and DH5⍺ were already readily available in our laboratory, Nissle 1917 was only available as a probiotic tablet (Mutaflor).
Figure 1. Whole genome sequence alignment of 3 different E. coli strains: DH5⍺ (orange); BL21(DE3) (purple); Nissle 1917 (grey) against the reference (known to attach to hair) ATC25922 (not shown).
Act I: Methods
In order to determine if the different E. coli strains did indeed adhere to hair we decided to transform our bacteria with constructs containing colour expressing genes. Initially we used the provided iGEM kit plate plasmids: we selected BBa_J23106 (mRFP1mRFP1 17O, 2019 iGEM kit plate; weak constitutive promoter) and BBa_I746909 (sfGFP 7M 2019 kit plate 3; T7 promoter). We also attempted to transform E. coli Nissle 1917. However, we were unable to transform this strain with the RFP construct required for testing. Hence, we only performed the attachment tests with BL21(DE3) and DH5⍺.
Hair sterilisation:
A 0.2 g hair sample derived from the distal part of the total hair length was obtained from two healthy donors (E and M). Donors had no dandruff, scalp diseases, or chemical treatments such as hair dyes, bleach, or perms.
After collection the hair samples underwent disinfection through autoclaving.
NOTE: Autoclaving was selected because this is an efficient, yet relatively gentle way of sterilisation: hair straighteners reach higher temperatures and hence we believe that the structure of the hair would not be damaged by this process. However, we also performed a disinfection cycle with another sample in parallel. This additional provided sample was held using an elastic band and placed in 70% ethanol on a previously autoclaved empty pipette tip box. The sample was left on a rocker for 5-10 minutes soaking on ethanol. Then, it was transferred to a new box containing sterile MilliQ water and rocked for a further 10 minutes, this step was repeated twice. Finally, the sample was left in fresh MilliQ water overnight with shaking and used the following day. Similar results were obtained following the disinfection cycle as well as those that had been autoclaved.
Bacterial sample preparation:
While the sample was being sterilised, E coli grown at 37ºC overnight and re-inoculated 50 µL of cells to 5 mL of fresh LB and their corresponding antibiotic (carbenicillin 50 µg/mL, or chloramphenicol 34 µg/mL). The bacterial inoculants were then incubated at 37ºC with shaking at 180 rpm. Once the desired OD600 ~0.5 had been reached, BBa_K199118 was induced with 50 µM of IPTG as it possesses a T7 promoter.
Sterile hair samples were placed in individual Eppendorf tubes to which 1000 µL of the induced bacterial culture was added. The hair samples were incubated with bacteria at 37ºC with shaking at 180 rpm and at different time points of 30 minutes, 1 hour and 2 hours they were retrieved and washed.
Three cycles of washings were then performed in sterile 0.9% sodium chloride (NaCl) solution. For each cycle, hair samples were placed in solution with NaCl and rocked for 10-15 minutes. After the washing cycle, samples were plated. Plating was performed with samples from different time points (30, 60 and 120 minutes) on agar plates containing the required antibiotic, when appropriate.
Act II: Results
Figure 2. Hair attachment was proven through the growth of our transformed coloured bacteria at different time points (30 minutes, 1 hour and 2 hours respectively). Then, the hair samples were washed and plated on agar plates with the required antibiotic chloramphenicol (sfGFP) and carbenicillin (mRFP1) and left incubating overnight at 37ºC. Plates are shown in different backgrounds: Top (black background) and bottom (white background) for visualisation purposes only. Two negative controls were performed: (washed hair) testing the sterilisation and (LB+hair) to ensure no contamination was obtained during the washing steps.
After having experimentally determined that E. coli DH5⍺ and BL21(DE3) attach to hair, we wanted to prove that our coloured constructs could also attach to hair. This coating would change the appearance of hair itself, hence providing the same effect as a commercially available temporary hair dye (Saitta et al., 2013).
Act III: Cutiful - into the wider world
We then decided to prove that transformed bacteria possessing our colour constructs were able to not only attach to hair, but also to produce colour.
Attachment was tested as above. However in this case bacteria were incubated with hair for only 30 minutes as our previous experiment has already proved that even in that short amount of time bacteria adherence occurred. Then hair samples were removed and placed on inducer-containing (anhydrotetracycline) agar plates and incubated for overnight growth.
Figure 3. Results show coating of natural, non dyed hair, with GM-coloured bacteria. Bacteria were transformed with green (BBa_K2906000), blue (BBa_K1455001), red (BBa_K092300). All 3 colour constructs were tested in both E. coli DH5⍺ as well as BL21(DE3); additionally, a negative control is shown (right) to show the lack of contamination during the process. Same results are shown over 3 different background for visualisation purposes: top (UV light), middle (black background) and bottom (white background).
This experiment shows that our coloured bacteria is able to coat hair samples and express the desired color. After already having determined that we had bacteria that would attach to hair, we talked with the Institute of Trichologists. During our interview with Liam Byrne (Institute of Trichologists), concerns about our bacteria’s continued survival on hair were brought up: can our bacteria survive in harsh conditions such as chlorinated swimming pools, saltwater of the ocean or normal shampoo washings? For our product to function we need our bacteria to survive in these conditions, at least to the extent where the colony can revive following treatment. Swimming is a common and popular activity, and so we decided to investigate the trichologists’ concerns and test our bacteria’s survival through harsh salt/chlorine conditions. Additionally, we also identified if our bacteria were able to resist washing with regular supermarket-available shampoos.
Salt and Chlorine
Experiment:
To test survival in various conditions we observed the effects on population size following a 2h incubation period in LB supplemented with sodium hypochlorite or salt. Bacterial stocks were initially pre-cultured overnight at 37°C in a shaking incubator then grown till reaching approximated OD600 = 0.5. These stocks were then split into flasks containing media representing the various growth conditions (LB control; hot tub chlorination - 3ppm NaClO; 2ppm NaClO; swimming pool chlorination - 1ppm NaClO; and sea salinity - 35g/L NaCl); these were then incubated for 2h at 37°C in a shaking incubator. Surviving populations were diluted, plated and incubated overnight for CFU counts the next morning. Each strain’s CFU count was analysed for differences between growth conditions using a one-way ANOVA using GraphPad Prism 8.2.1. and a significance threshold of p<0.05. When significant differences were detected a post-hoc Dunnett’s multiple comparisons test was used to compare growth conditions to the positive control as to identify which treatments were significant.
Beyond survival we also want our bacteria to keep expressing colour following this treatment. To find out whether this would be realistic, we tested both our RFP-expressing DH5⍺ and BL21(DE3) bacteria while carrying out the CFU count on inducer plates. The use of inducer plates allowed us to visually confirm the production of RFP following salt or choline treatment based on the colour of colonies. This allowed us to confirm that following our incubation our bacteria would still be able to produce our colour proteins.
Results:
In all scenarios, large populations of red bacteria remained viable after treatment suggesting that our product will not be lost via exposure to chlorinated water (1-3ppm) or sea water (35g/L NaCl). Additionally, neither DH5⍺ nor BL21(DE3) showed significant differences in growth following hypochlorite treatment (t-test P>0.05). Interestingly, BL21(DE3) did show significant growth impairment from the addition of 35g/L NaCl (t-test P<0.05) whereas DH5⍺ showed no significant effect, this may imply that BL21(DE3) is more sensitive to high salt levels than DH5⍺ (Figure 4).
Figure 4. The Survival of BL21(DE3) & DH5⍺. Graphs show mean colony forming units and standard deviation recorded after 2h incubation in LB and LB supplemented with Hypochlorite or salt. All graphs demonstrate bacterial survival in all conditions. The 35g/L NaCl LB significantly reduced BL21(DE3) survival (t-test P<0.05) however all other conditions showed no significant difference. DH5⍺ tests on the whole showed more apparent variance, however, still confirm bacterial survival. * indicates significance (t-test P<0.05)
Our DH5⍺ results show more apparent variance than the BL21(DE3) results, BL21(DE3) grows faster than DH5⍺, and so the reduced number of bacteria may result in increased variance in dilution prior to plating which then would translate into the count.
It is also surprising how insignificant the effect of chlorination was. In swimming pools and hot tubs shock chlorination is used where one add a large amount of hypochlorite then lets it reduce to 1-3ppm before declaring it safe for human usage. As a result, it is possible that the majority of bactericidal action occurs at that shock stage. Another possibility is an error in experimental method: each incubation occurred in LB which is a very rich medium. It is possible that the active chlorine initially added reacted with the LB and so the true concentration of active chlorine was lower during incubation. Therefore, it could be useful for further studies to determine the actual amount of active chlorine in the medium, or to perform the two-hour exposure in a buffered aqueous solution instead of a full growth medium.
Shampoo Exposure
After having identified that our bacteria would not only attach to hair but also remain viable under daily life activitird such as swimming in chlorinated or salt-containing waters we decided to ensure if they would be resistance to washing with shampoo.
Experiments:
Hair samples were incubated with our mRFP1 construct as above. However, in this case they were retrieved after 30 minutes. After recollection, the samples were washed with 0.9% NaCl followed by a thorough wash in two different shampoo types (referred to as Shampoo 1 and 2 respectively). The shampoos were diluted 1 in 20 to simulate normal showering conditions. Then they were rinsed as above in two cycles of saline water with rocking for 10-15 minutes each time. The hair samples were then placed on agar plates with anhydrotetracycline inducer and incubated overnight at 37ºC.
Figure 5. Results are seen both in white background (top image) and black background (bottom image). Negative control was performed through the plating of LB with Hair; positive control contains hair samples that have only been washed in NaCl and lastly both shampoo 1 and 2 have an additional washing step in diluted Shampoo.
From our results we can infer that our product would not be lost by washing with a standard shampoo. This means that the dye would be semi-permanent and would require safety measures. Therefore, it would require additional experiments to show the amount of time that the hair dye would work.
Modular and Personalisable
This page provides a successful demonstration of the modular nature of our product as not all three secreted compounds need to be transformed into a single bacteria. The ability to mix an interchange the parts allows a customisable product. We learned through our public outreach that not everyone will want to colour their hair, however, they may be interested in the repair and scent portion of the project. Hence, the division of our compounds into different cells allows the customer with a power of decision. We have additionally demonstrated that the mixing of different ratios of coloured bacteria (through the use of co-cultures) allow the formation of a whole spectrum of colours, as requested by end users (Human Practices, Act II: The Community Festival: Act V: Conversations with Experts).
Epilogue: Future Experiments
Since transformation of Nissle 1917 was not achieved, we believe that further experimentation should follow this path. The significance of an engineered E. coli Nissle relies on its probiotic properties: we believe that it would be more easily accepted by customers than other tested strains. We expect that the adherence of this bacterial strain would be just as good as that of the tested strains, as suggested in Figure 1, which shows high genome similarity with the ATCC 25922 E. coli strain known to be part of the natural hair microbiome.
We demonstrate that bacteria would not be lost from hair in response to washing with standard shampoo currently available on the market. While this is a desirable result, it also has implications for the safe disposal of our product. Therefore, future experiments incorporating the modelled kill-switch would be required. Additionally, other experiments should test if bacteria are also resistant to other daily hair beauty products such as conditioners, hair gels, dry shampoo, etc. Additionally, in the future we would like to test it on bleached hair: this way we can move forward with the feasibility of the dye by testing colour retention over a period of time.
Our vision would be to supply a complete all-in-one product able to care and manipulate hair by offering a wide range (Human Practices, Act V: Conversations with Experts) of colours, fragrances, structure and repair components using a bacterial whole cell system. This would allow us to overcome the problems caused by current marketed hair products (see project description).