As part of our efforts to create a platform for easier design of kill switches, our experimental team has characterised new iGEM parts and improved parts to suit potential users who are seeking customised kill switches for containment of engineered probiotics.

Our human core temperature lies in the range of 36 – 37 degree Celsius. This makes temperature a good physical stimulus for us to leverage upon when designing a kill switch to work outside a human body. We have utilised the thermal sensitive protein TlpA36's capability of dimerising at temperatures higher than 36 degree Celsius, which makes it unable to bind to its intrinsic promoter pTlpA. This system can form a temperature-sensitive switch to sense the temperature drop when the engineered probiotic escapes from the warmer human host.

As an illustration, by coupling the temperature system to a GFP reporter BBa_K2447014, under a temperature of 36 degree Celsius or higher (‘ON’ switch condition), GFP production will not be significantly repressed. However, when the temperature drops below 36 degree Celsius (‘OFF’ switch condition), GFP production will be repressed.

We have put the TlpA36 protein under the control of a pLac promoter to check the TlpA36 concentration effect on GFP expression. For 37 degree Celsius, the GFP expression falls with an increasing concentration of IPTG. On the other hand, at 30 degree Celsius, the increasing IPTG concentration seems to have minimal impact on the GFP repression. It is a notable fact, that the sensor behaves best without any IPTG in both cases, which suggests that little TlpA36 is required for sensor activation.

Transformed 10β cells were incubated with shaking in LB broth with kanamycin (50 ng/µL) at 30 degree Celsius for 48 hours, before being subcultured 1:100 and then incubated for another 2-3 hours to reach an OD¬600nm of 0.1. Next, cells were loaded into a 96-well plate containing various concentrations of IPTG. A 10-min interval reading of OD¬600nm and GFP absorbance was conducted over a continuous 8 hours run of the microplate reader at a temperature of 30 or 37 degree Celsius.

Figure 1: BBa_K2447014 Temperature-system coupled to GFP reporter. At 37 degree Celsius, the system is switched ‘ON’, generating a 1-fold increase in GFP production.

Phosphate ions contain phosphorus which is an important component for healthy teeth and bone. Hence, our body readily absorbs these vital ions in our gut. The low phosphate concentration in the intestines following absorption earlier in the tract, is thus an important chemical stimulus for us to leverage upon in the design of a kill switch, which is able to detect changes in the host’s environment as the engineered probiotics travel out of the human body. Having guided by in silico modelling, we have come up with a phosphate-temperature cascaded system.

As a first step, we have layered the phosphate-sensitive promoter over the temperature sensitive system and coupled it to a GFP reporter BBa_K2447015. This part was designed in such a way that the temperature sensitive protein TlpA36 is only produced when the phosphate concentration is minimal (to simulate that the engineered probiotic has reached the gut region). However, before exiting the human environment, the temperature is still high enough (at 36 degrees and higher), for TlpA36 proteins to remain as dimers and thus GFP production is not repressed. However, once the engineered probiotic leaves the human GI tract, the temperature drops below 36 degrees; thus TlpA36 proteins dimerise and bind onto its promoter tlpa36 and repressing downstream GFP expression.

Transformed 10β cells were incubated in LB broth with kanamycin (50 ng/µL) at 30 degrees for 48 hours before being diluted 100x and then incubated for another 2-3 hours to reach an OD of 0.1. Cells are washed in MOPS medium (with 0.2% casamino acid and 0.2% glucose) and subsequently re-suspended in MOPS medium (with 0.2% casamino acid and 0.2% glucose). Next, cells are loaded into 96 well plate preloaded with various concentrations of phosphate concentrations. 10 mins interval reading of OD¬600 and GFP absorbance was conducted over a continuous 8 hours run of the microplate reader at temperatures of 30 and 37 degrees (separately).

Figure 2: Bba_K2447015 Phosphate-temperature sensitive cascaded system with GFP reporter. At 37 degree, the engineered probiotic still remain in the human body and hence GFP production is turned ‘on’. At 30 degree, the probiotic is assumed to be out of the human system, and hence GFP production is turned ‘off’.

Following the feasibility of the previous construct Bba_K2447015 in showing GFP production at the appropriate phosphate and temperature stimulus, we have taken the final step into the design of the two-inputs kill switch. This cascaded system BBa_K2447017 has replaced GFP with E2 Immunity protein (IM2, and a further downstream constitutive E2 Colicin protein (a type of exonuclease). Following the same circuit logic as Bba_K2447015, IM2 proteins will be constantly produced when the engineered probiotics is still inside the human body; thereby able to sequester E2 killing protein and keeping the engineered probiotics alive. However, once it is out of the human body, IM2 protein production will be repressed, causing E2 killing protein to accumulate in the bacterial cell thereby degrading its DNA and breaking down the cell membrane.

(to be updated)

Figure 3: Bba_K2447017 Phosphate-temperature cascaded kill switch circuit design. High phosphate concentration would allow constant IM2 protein production (kill switch not activated). Only at low phosphate concentration and at lower temperature (less than 36), IM2 production would be repressed and unable to sequester E2 killing protein. Hence the kill switch is activated.

Figure 4: Bba_K2447017 Cell death characterisation at 37 degree and 30 degree. Under low phosphate level and low temperature, cell viability is significant lower due to repressed IM2 production and over accumulation of E2 killing protein.

Apart from using temperature and phosphate levels, our team had thought of using blue light as a stimulus for our kill switch application. Since the engineered probiotics would be eventually excreted out of the human body after consumption, modern toilet seats could be slightly altered to shine blue light on the faeces, to switch the kill switch ‘ON’.

Figure 5: Modern toilet seat could be modified to include blue light LEDs to activate the kill switches in the engineered probiotics. Source: https://www.aliexpress.com/item/Sensor-Toilet-Light-8-Colors-LED-Battery-operated-Lamp-lamparas-Human-Motion-Activated-PIR-Automatic-RGB/32779033584.html

To illustrate the feasibility of such design, our team had constructed the blue light inducible system with RFP reporter BBa_K2447501. In this circuit, EL222, a bacterial photosensitive protein is constitutively produced. When the cells are shone with blue light, EL222 proteins dimerise and bind upstream of the luxI promoter, allowing the recruitment of RNA polymerase, followed by downstream expression of RFP. When light is absent, EL222 proteins are not activated, and hence there is minimal RFP expression. Using this part as an example, the kill switch would be turned ‘ON’ when blue light is shone. Under blue light illumination, both wild type cell (MG 1655) and 10 cells exhibited 3 times increase in RFP expression when compared to no light condition, by the 6th hour.

ransformed MG 1655 and beta 10β cells are incubated in LB broth broth with kanamycin (50 ng/µL) at 37 degrees for 24 hours before being diluted 100x and then incubated for another 2-3 hours to reach an OD of 0.1. Next, cells are loaded onto 12 well plate. Hourly readings of RFP and OD600 were taken and the cells were illuminated with blue light (control group: covered with black cloth) for 7 hours consecutively under orbital shake (120 rpm) and 37 degrees.

Figure 6: BBa_K2447501 Blue light inducible RFP expression. Source: https://www.ncbi.nlm.nih.gov/pubmed/27353329

Figure 7: BBa_K2447501 Blue light inducible system with RFP reporter. Transformed cells are strongly switched ‘ON’ when blue light is shone as observed by the 3 fold increase in RFP expression when compared to no light condition. Both MG 1655 and 10β cells have reported similar RFP expressions.