iGEM 2017 — Team NUS_Singapore

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# Team:NUS Singapore/Notebook

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[Collaboration](https://2017.igem.org/Team%3ANUS_Singapore/Collaborations)

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Modelling

[Overview](https://2017.igem.org/Team%3ANUS_Singapore/Overview)
[Methodology](https://2017.igem.org/Team%3ANUS_Singapore/Methodology)
[Kill Switch for Probiotics](https://2017.igem.org/Team%3ANUS_Singapore/Model_P)
[Kill Switch for BeeT](https://2017.igem.org/Team%3ANUS_Singapore/Model_B)

Projects

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# Lab Journal

![](https://static.igem.org/mediawiki/2017/a/a1/NUS_2017_IGEM_may.png)

May

## First three weeks

Brainstorming for potential ideas. The team have considered a few different topics including detection of sound wave with microbes and design of urinary catheter antimicrobial microbes. After intensive reading and research, the topic was crystalized to the design of a kill-switch for engineered probiotics application.

As part of our safety culture and practices in NUS, we went on to attend an online safety course and successfully obtained lab safety certificates: A series of online modules to fulfil safety requirements by Office of Safety, Health and Environment (OSHE, http://www.nus.edu.sg/osh/), the department in charge of Laboratory and Work Safety at the National University of Singapore.

Our team also attended the safety training events before being able to conduct lab work in Centre for Life Science (CeLS), NUS.

## Last week

Basic hands on experiments with the entire construction process. Grasp essential lab skills needed for iGEM such as PCR, Gibson assembly and transformation.

We began Interlab Study: Cell transformation was performed. Unfortunately, very low transformation efficiency was observed.

![](https://static.igem.org/mediawiki/2017/0/0a/NUS_2017_IGEM_JUNE.png)

June

## First Week

Continuation of Interlab Study: Identifying possible causes of failure (e.g. cells were perhaps left in the ice for too long or cell lysis may occur during prolonged mixing) The transformation process was repeated. The team also completed the third part of the Interlab Study where we conducted cell density measurement at 0, 2, 4, 6, hours, with a micro plate reader.

## Second Week

Design of the plasmids that will be used in the project, Subsequently required gblocks were ordered from IDT.

## Third Week

The whole team had participated in the Seventh International Meeting on Synthetic Biology (SB7.0). We had gained insights and met researchers from different parts of the world.

For the experiments, we had started construction of the phosphate-sensor with GFP reporter (our improved part, pGEMB), the temperature-sensitive GFP reporter system (our new part, pGEME) and inducible E2 killing protein expression system (pGEMF).

Here is a brief description of the steps:

* PCR:

1. Prepare the master mixture. 3) PCR for 1:30 hours in the PCR machine (30 cycles).
2. Add forward and reverse primers for the constructs.
3. PCR for 1:30 hours in the PCR machine (30 cycles).

* Gibson Assembly:

1. Prepare the mixture of DNA backbone and respective gblocks with Gibson Master Mix.
2. Incubate in 50 degrees for a total of 0.5 hours in the thermocycler.

* Cell transformation:

1. Mix competent cells with assembled plasmid mixture.
2. Heat shock for fifty seconds and cool down for three minutes.
3. Add LB broth.
4. Incubate in the shaking incubator for one hour.
5. Place the agar plate in the 37-degrees incubator for one hour.
6. Plate the cells.

* Cell inoculation:

+ Two tubes (6-7 ml of LB broth). Inoculate each tube with one colony.

* Preparing glycerol stock:

+ 850 µL of solution from cell inoculation into glycerol solution and keep at -80-degrees fridge for long term storage.

* Plasmid extraction - Follow the protocol in the manual.
* Sequencing:

1. Prepare sequencing primer.
2. Primer and 2 samples of constructed plasmid were sent for sequencing.

## Last Week

We had received sequencing results and the results were not satisfactory. Construction of phosphate sensor with GFP reporter was done again.

![](https://static.igem.org/mediawiki/2017/e/ea/NUS_2017_IGEM_july.png)

July

## First Week

Sequencing result for the phosphate sensor with GFP reporter was good. MOPS medium was purchased for future characterisation of our phosphate sensor-GFP reporter.

We have began second try of reconstruction for temperature sensitive system - with GFP reporter, and reconstruction of E2 expression system.

## Second Week

Sequencing result for temperature sensitive system was good. We could begin characterisation of this system in the next few weeks.

MOPS minimal medium and phosphate stock solution were prepared. Plasmid containing the phosphate sensor was retransformed into MG 1655 for characterisation with a microplate reader next week.

## Third Week

8 hours (10 mins sampling of OD and GFP) microplate reading was conducted for the phosphate sensor-GFP reporter. From the characterisation results, the phosphate sensor looked promising, exhibiting greater GFP production at lower concentrations of phosphate, while repressing GFP production at high phosphate concentrations.

Construction of E2 killing system was repeated this week and the plasmid was sent for sequencing.

## Last Week

The plasmid with temperature sensitive system (with GFP reporter) was re-transformed into MG 1655. 8 hours of continuous micro plate reading was conducted at 37 degrees to gauge the GFP production of the wild type cells under varying IPTG levels.

Construction of the killing system was still not successful, primers for this system were redesigned. Nevertheless, we had managed to complete two constructs (temperature system and phosphate sensor) in 2 months.

![](https://static.igem.org/mediawiki/2017/d/d9/NUS_2017_IGEM_aug.png)

August

## First Week

Re-characterisation of phosphate system (in MG1655) was done to verify the GFP results.

Start of school semester.

Characterisation of temperature sensitive system (MG 1655) was conducted at 30 degrees. Results did not look promising, GFP expression at lower temperature is much higher than GFP expression at higher temperature.

Concurrently, initial and final 24th hour plate reading of the overnight cultures at 30 and 37 degrees (with the temperature system) was conducted. Surprisingly and Interestingly, the GFP results obtained were as expected, where there is higher GFP production at higher temperature.

Construction of the killing system is still not successful. Construction was again repeated, with an optimised gblock to backbone ratio.

## Second Week

Began construction of phosphate-temperature cascaded system with GFP reporter. Characterisation of this construct was conducted with varying phosphate concentrations under two different temperatures, 30 and 37 degrees. Again, the GFP results does not look promising, showing greater GFP expression in lower temperatures.

Sequencing results for killing system was not good. Began characterisation for this system, and as expected, results were not promising, as the MG 1655 cells had died too quickly, perhaps due to high leakiness of the system to produce E2 killing protein even in repressed states. As an alternative, we had redesigned the killing plasmid with a tighter TetR system. Construction of this system had begun this week.

## Third Week

Characterisation of new TetR based killing system. However, the transformed MG 1655 cells still die too quickly.

## Last Week

Decided to stop experimenting on the killing system and start focusing on ‘de-bugging’ the temperature sensitive system which performs in opposite fashion to what was expected.

In response, we had designed the thermal gradient assay by using a conventional thermal cycler to set varying temperature gradient from 30 to 37 degrees across the rows. GFP reading and OD600 were obtained at the 0 hour and 24th hour.

![](https://static.igem.org/mediawiki/2017/c/c9/NUS_2017_IGEM_sep.png)

September

## First Week

Repeated the same thermal gradient protocol as last week. To reduce liquid loss impact during the culture transfer,we had loaded 100ul of cells instead of 50ul into each wells and an initial and final plate reading (at the 24th hour) would only be taken.

This is the detailed plan: Culture the transformed cells ( the temperature sensitive system construct, is transformed into MG 1655) from glycerol and incubate for 36 h in 30 degrees in LB medium; take 100x dilution of the original sample and regrow in fresh LB medium for 4 hour; load 100µL of cell into each well of the transparent plate and do initial microplate read (0 hr reading) before transferring to PCR plate for incubation; after incubating for 24 hours, transfer from PCR plate to transparent 96 wells plate for plate reading (at 24hours time point ).

## Second Week

Construction of nitrate sensor with GFP reporter and phosphate sensor with GFP reporter (weaker RBS BBa\_B0032) had began.

Characterisation of temperature system (MG 1655) in MOPS medium with2mM of phosphate ions. Characterisation of the phosphate-temperature cascaded system (MG 1655) was also conducted in MOPS medium, with varying amounts of phosphate ions. .

## Third Week

The sequencing results for the modified nitrate and phosphate sensor were good, so we begun transformation and characterization this week (the nitrate protocols can be found here: http://parts.igem.org/Part:BBa\_K381001:Experience). We re-characterised the temperature-sensitive system and also the phosphate-temperature system at higher starting OD600 of 0.3 or 0.4a plasmid containing constitutive GFP production was used as a control.

## Last Week

Characterisation results of the temperature system and phosphate-temperature system Were not in line with expectations.

We re-transformed the plasmid (temperature sensitive system) into 10β cells to compare its GFP expression with earlier results from MG 1655. Characterisation of temperature sensitive system with the thermal gradient assay showed the expected results, which yielded higher GFP production at higher temperatures.

Characterisation of both phosphate sensors (one is with RBS BBa\_B0034 and one is with RBS BBa\_B0032) in MOPS medium under varying phosphate concentrations. Characterisation results were satisfactory and suggested the phosphate sensor with stronger RBS had showed 40 folds increase in GFP expression when compared to the other phosphate sensor construct.

![](https://static.igem.org/mediawiki/2017/e/eb/NUS_2017_IGEM_oct.png)

October

## First Week

To prepare submission of standard parts in pSB1c3 to iGEM HQ, we have constructed rrnB-EL222 with this backbone and transformed into 10β cells. This construct will constitutively express EL222 proteins, which is a blue-light sensitive transcription factor.

## Second Week

Casamino acids assay was conducted to characterize the effects in affecting the phosphate sensor in phosphate-temperature system (10β).

We characterised the construct with temperature sensitive system (10β) and the construct with phosphate-temperature system (10β) under respective inducers of IPTG and phosphate in MOPS with 0.2% casamino acids at 37 degrees as well as in 30 degrees.

Phosphate-temperature cascaded system with controlled expression of IM2 (E2 immunity protein) was constructed and transformed into 10β.

Blue light inducible system with RFP expression was transformed into MG 1655 and 10β. Characterisation of RFP over 8 hours under blue light and control conditions (no blue light) was conducted.

## Third Week

Construction of pGEMBECF (phosphate-temperature inducible Im2 expression and constitutive E2 production) had begun.

CF-Ind (blue light activating RFP production) in LB media for 10β and MG 1655 (with orbital shaking at 120rpm) was conducted. Under blue light illumination, EL222 photosensitve protein (constitutively expressed) is binded upstream of the luxI promoter, enabling RNA polymerase to bind and trigger RFP expression. Without blue light illumination, EL222 protein cannot bind.

pGEMBE (phosphate-temperature cascaded system) construct characterisation (at higher starting OD600 = 0.2) in MOPS and varying phosphate levels was conducted.

PGEMBECF OD600 – cell death characterisation was conducted.

Construction of TlpA36-psb1c3 standard backbone was done.

Construction of EL222-psb1c3 standard backbone was done.

## Last Week

Characterisation of pGEMBE(phosphate-temperature cascaded system) in MOPS medium under two different temperature conditions ( 37 and 30 degrees), a 8 hour continuous plate reading is conducted where GFP and OD readings are obtained every 10mins.

In addition, for this construct pGEMBE, under incubation of various temperatures (30 to 37 degrees), GFP production is obtained for every cell by using the flow cytometer.

Characterisation of cell death profile of pGEMBECF (phosphate-temperature inducible Im2 expression and constitutive E2 production) in MOPS medium under two different temperature conditions ( 37 and 30 degrees), a 8 hour continuous plate reading is conducted where OD readings are obtained every 10mins.

Miniprep and dry down of the 3 plasmids [(A) rrnB-EL222-PSB1C3, (B) Tlpa36 standard-PSB1C3 & (C) Phosphate-rbs34-PSB1C3 ] to be shipped over to iGEM headquarters.

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# Team:NUS Singapore/Experiments

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Awards Check

[Measurement](https://2017.igem.org/Team%3ANUS_Singapore/Measurement)
[Modelling](https://2017.igem.org/Team%3ANUS_Singapore/Model)
[Medal Check List](https://2017.igem.org/Team%3ANUS_Singapore/Medal%20Check%20List)

Team

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[Attributions](https://2017.igem.org/Team%3ANUS_Singapore/Attributions)

Collaboration

[Collaboration](https://2017.igem.org/Team%3ANUS_Singapore/Collaborations)

Human Practice

[Silver Up](https://2017.igem.org/Team%3ANUS_Singapore/HP/Silver)
[Gold Integrated](https://2017.igem.org/Team%3ANUS_Singapore/HP/Gold_Integrated)

Modelling

[Overview](https://2017.igem.org/Team%3ANUS_Singapore/Overview)
[Methodology](https://2017.igem.org/Team%3ANUS_Singapore/Methodology)
[Kill Switch for Probiotics](https://2017.igem.org/Team%3ANUS_Singapore/Model_P)
[Kill Switch for BeeT](https://2017.igem.org/Team%3ANUS_Singapore/Model_B)

Projects

[Description](https://2017.igem.org/Team%3ANUS_Singapore/Description)
[Interlab Study](https://2017.igem.org/Team%3ANUS_Singapore/InterLab)
[Parts](https://2017.igem.org/Team%3ANUS_Singapore/Parts)
[Experiment](https://2017.igem.org/Team%3ANUS_Singapore/Experiments)
[Documentation](https://2017.igem.org/Team%3ANUS_Singapore/Notebook)
[Improvement](https://2017.igem.org/Team%3ANUS_Singapore/Improvement)
[Demonstrate](https://2017.igem.org/Team%3ANUS_Singapore/Demonstrate)
[Safety](https://2017.igem.org/Team%3ANUS_Singapore/Safety)
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# Experiment

## Overview

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

## New Part 1: Temperature Sensitive System **✔Fully Functional**

Our human core temperature lies in the range of 36 – 37 °C. This makes temperature a good physical stimulus for us to leverage upon when designing a kill switch to work inside a human body. We have utilised the thermal sensitive protein’s TlpA36 capability of de-dimerising at temperatures higher than 36 °C, 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 had escaped from the human host.

As an illustration, by coupling the temperature system to a GFP reporter  [BBa\_K2447014](http://parts.igem.org/Part%3ABBa_K2447014), under temperature of 36 °C and higher (‘ON’ switch condition), GFP production will not be significantly repressed. However, when temperature is below 36 °C (‘OFF’ switch condition), GFP production will be repressed.

We have put the TlpA36 protein under control of pLac promoter to check the TlpA36 concentration effects on the GFP expression. For 37 °C (Figure 1), the GFP expression falls with the increasing concentration of IPTG. On the other hand, in 30 °C the increasing IPTG concentration seems not to have much impact on the GFP repression. It is a notable fact, that sensor behaves in both cases best without any IPTG, which would suggest that minimum TlpA36 is required for sensor activation.

**Characterisation of the temperature construct had proven to be challenging for the experimental team** as the temperature control behaves differently in each individual *E.coli* strain. Parallel studies involving both transformed 10β and MG1655 cells were conducted to understand whether the temperature construct would be functional in different strains. In this study, (Figure 2 and 3) GFP readings were obtained after 24 hours of incubation. This temperature sensitive construct was working as expected in 10β cells, elucidating higher GFP expression at higher temperatures. However, it was not functioning properly in MG1655, where the GFP expressions were much higher at lower temperatures.

### Characterisation Protocol:

Transformed 10β cells were incubated in LB broth with kanamycin (50 ng/µL) at 30 °C for 48 hours before being diluted 100x and then incubated for another 2-3 hours to reach an OD600 of 0.1. Next, cells were loaded into 96 well plate preloaded with various concentrations of IPTG. 10 mins interval reading of OD600 and GFP absorbance was conducted over a continuous 8 hours run of the microplate reader at temperatures of 30 °C and 37 °C (separately).

![](https://static.igem.org/mediawiki/2017/0/0d/NUS_IGEM_2017_exp001.png)

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.

![](https://static.igem.org/mediawiki/2017/d/d1/NUS_IGEM_2017_exp002.JPG)

Figure 2: BBa\_K2447014 construct which was characterised in 10β cells (incubated overnight). At higher temperatures, the system is switched ‘ON’ as seen by the increase in GFP expressions.

![](https://static.igem.org/mediawiki/2017/2/25/NUS_IGEM_2017_exp003.JPG)

Figure 3: BBa\_K2447014 construct which was characterised in MG1655 cells (incubated overnight). Unlike the construct in 10β cells, these wild type cells do not exhibit the proper ‘ON’ switch state at higher temperatures; where GFP production is expected not to be repressed.

## New Part 2: Phosphate-temperature Cascaded System **☒partially working, needs improvement**

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](http://parts.igem.org/Part%3ABBa_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 °C and higher), for TlpA36 proteins to exist as monomers and thus GFP production is not repressed. However, once the engineered probiotic leaves the human GI tract, the temperature drops below 36 °C; thus, TlpA36 proteins can dimerise and bind onto its pTlpA promoter, repressing downstream GFP expression.

From our experiment (Figure 4), we had successfully elucidated 1-fold higher GFP expressions at the higher temperature (37 °C) when compared to the GFP expressions at 30 °C. This confirmed the fact that our temperature sensitive system (consisting of TlpA36 and pTlpA) was fully functioning, creating a repressible effect on downstream gene expression, only when temperature was below 36 °C. However, this layered construct was not working as expected in terms of sensing for the presence of extracellular phosphate ions. Regardless of the temperature conditions, at high phosphate concentrations, GFP expressions were expected to be similar, but we had obtained differing GFP productions at two different temperatures (37 °C and 30 °C) even at the same phosphate concentration. This may be due to the fact of cell metabolism being significantly different in 30 °C and 37 °C. More importantly, the phosphate promoter might not be tight enough to prevent TlpA36 from leaking. In short, our phosphate-temperature construct was working to a large extent, where the temperature control was functionally properly. But due to time constraint, we did not have the chance to rectify the issue of leakiness in the phosphate promoter, possibly by optimising the binding affinity of this promoter.

### Characterisation Protocol:

Transformed 10β cells were incubated in LB broth with kanamycin (50 ng/µL) at 30 °C for 48 hours before being diluted 100x and then incubated for another 2-3 hours to reach an OD600 of 0.1. Cells were 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 were loaded into 96 wells plate preloaded with various concentrations of phosphate. To characterise cell death, readings of OD600 with 10 min interval were conducted over a continuous 8 hours run in the microplate reader at temperatures of 30 °C and 37 °C (separately).

![](https://static.igem.org/mediawiki/2017/e/e8/NUS_IGEM_2017_exp004.png)

Figure 4: Bba\_K2447015 Phosphate-temperature sensitive cascaded system with GFP reporter, experimentation with a microplate reader for continuous 8 hours incubations at both 37 °C and 30 °C. At 37 °C, the engineered probiotic remains in the human body and hence GFP production is turned ‘ON’. At 30 °C, the probiotic is assumed to be out of the human system, and hence GFP production is turned ‘OFF’.

## New Part 3: Phosphate-temperature Cascaded Kill Switch **☒needs further experimentation**

Following the feasibility of the previous construct ([BBa\_K2447015](http://parts.igem.org/Part%3ABBa_K2447015)) in showing GFP production at the appropriate phosphate and temperature stimuli, we have taken the final step into **the design of the two-inputs kill switch, which is consisted of a double plasmid system**. **In the first plasmid, the double-inputs cascaded system ([BBa\_K2447016](http://parts.igem.org/Part%3ABBa_K2447016)) would control IM2 anti-toxin production (a type of E2 Immunity protein, to prevent cell death).** This anti-toxin plasmid will work in tandem with **the second plasmid ([BBa\_K2447017](http://parts.igem.org/Part%3ABBa_K2447017)), a standardised plasmid that is constitutively producing E2 killing protein (Colicin, a type of endonuclease).**  However, due to time constraint, we did not construct the standardized plasmid.

Following the same circuit logic as the phosphate-temperature cascaded system with GFP reporter ([BBa\_K2447015](http://parts.igem.org/Part%3ABBa_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.

Earlier experimentation with E2 killing protein had proven to be highly challenging due to, most probably, E2 proteins high efficacy in killing the cells, even if the IM2 protein was expressed at the same time. This can be attributed to overproduction of E2 from the medium strength constitutive promoter that we tried to put the protein under. In consequence, our transformation plates were usually empty due to all the transformed cells being killed upon receiving the plasmid.

Nevertheless, even without the actual construct, our team had modelled this kill switch in AdvanceSyn platform to provide insights of IM2 protein production at various phases with different phosphate levels and temperature conditions (to simulate the bacteria moving down the digestive tract and eventually escaping the body). By manipulating the relative promoter strength of the phosphate promoter via changes in its Vmax (Figure 6), we have observed an uncalled decline in IM2 production, which would prematurely kill the cells due to accumulation of E2 killing protein. This had coincided with the results of our previous experiments with the phosphate and temperature cascaded system with GFP reporter (BBa\_k2447015). This highlighted that the phosphate promoter was leaky in its repressed state (at high phosphate concentration) and it may be possibly due to the relatively high binding strength of the phosphate promoter, as pointed out by the model (Figure 6); leading to over production of unwanted TlpA36 proteins. Subsequently, we had modelled (Figure 7) the effects of the pTlpA promoter on IM2 production, while constitutively expressing the E2 killing protein to reveal the IM2-E2 protein interaction. This model (Figure 7) had shown that the promoter pTlpA must be of higher binding strength (higher Vmax) to show sufficient downstream IM2 expression to sequester the constitutively expressed E2 killing protein.

Based on the modelling insights, future attempts of improving the kill switch could include decreasing the binding affinity of the phosphate promoter and increasing the binding affinity of the downstream temperature sensitive promoter (preferably both). This change in the affinities could be done with use of directed mutagenesis. However, due to the difficulties in engineering the promoter, changing the RBS strength used for the given protein expression could be preliminary solution to optimise the balance between the IM2 and E2 proteins.

### Characterisation Protocol:

Co-Transformed 10β cells (with double plasmid kill switch system) were incubated in LB broth with kanamycin (50 ng/µL) at 30 °C for 48 hours before being diluted 100x and then incubated for another 2-3 hours to reach an OD600 of 0.1. Cells were 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 were loaded into 96 wells plate preloaded with various concentrations of phosphate. To characterise cell death, readings of OD600 with 10 min interval were conducted over a continuous 8 hours run in the microplate reader at temperatures of 30 °C and 37 °C (separately).

![](https://static.igem.org/mediawiki/parts/c/c9/Doubleplasmid.png)

Figure 5: Double plasmids kill switch system consisting of Bba\_K2447016 (controlled anti-killing IM2 protein) and Bba\_k2447017 (constitutive E2 killing protein production).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 °C), IM2 production would be repressed and unable to sequester E2 killing protein. Hence the kill switch would be activated.

![](https://static.igem.org/mediawiki/2017/2/2e/NUS_IGEM_2017_exp006.png)

Figure 6: Modelling of the IM2 production by the phosphate promoter in our proposed kill switch. The strength of the phosphate promoter were altered (by changing the Vmax). Higher Vmax for this promoter had elucidated issues of leakiness in the system.

![](https://static.igem.org/mediawiki/2017/d/d9/NUS_IGEM_2017_exp007.png)

Figure 7: Modelling of the temperature sensitive promoter, pTlpA which controls IM2 productions in our proposed kill switch. To find an ideal balance between IM2 and E2 levels (assuming E2 protein interact with IM2 protein in 1 to 1 ratio), the strength of the temperature promoter was altered (by changing the Vmax). Higher Vmax for this promoter had been thought to be ideal to produce sufficient IM2 to sequester the E2 killing proteins.

## New Part 4: Blue-light Inducible System with RFP Reporter **✔Fully Functional**

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’.

![](https://static.igem.org/mediawiki/2017/0/00/NUS_2017_IGEM_experiment005.png)

Figure 8: Modern toilet seat could be modified to include blue light LEDs to activate the kill switch 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](http://parts.igem.org/Part%3ABBa_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.

### Characterisation Protocol:

Transformed MG 1655 and 10β cells are incubated in LB broth with kanamycin (50 ng/µL) at 37 °C for 24 hours before being diluted 100x and then incubated for another 2-3 hours to reach an OD600 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 °C.

![](https://static.igem.org/mediawiki/2017/7/76/NUS_2017_IGEM_experiment006.png)

Figure 9: BBa\_K2447501 Blue light inducible RFP expression. Source: https://www.ncbi.nlm.nih.gov/pubmed/27353329

![](https://static.igem.org/mediawiki/2017/2/20/NUS_2017_IGEM_experiment007.png)
![](https://static.igem.org/mediawiki/2017/1/1c/NUS_2017_IGEM_experiment008.png)

Figure 10: 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.

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# Team:NUS Singapore/Results

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Awards Check

[Measurement](https://2017.igem.org/Team%3ANUS_Singapore/Measurement)
[Modelling](https://2017.igem.org/Team%3ANUS_Singapore/Model)
[Medal Check List](https://2017.igem.org/Team%3ANUS_Singapore/Medal%20Check%20List)

Team

[Team Member](https://2017.igem.org/Team%3ANUS_Singapore/Team)
[Attributions](https://2017.igem.org/Team%3ANUS_Singapore/Attributions)

Collaboration

[Collaboration](https://2017.igem.org/Team%3ANUS_Singapore/Collaborations)

Human Practice

[Silver Up](https://2017.igem.org/Team%3ANUS_Singapore/HP/Silver)
[Gold Integrated](https://2017.igem.org/Team%3ANUS_Singapore/HP/Gold_Integrated)

Modelling

[Overview](https://2017.igem.org/Team%3ANUS_Singapore/Overview)
[Methodology](https://2017.igem.org/Team%3ANUS_Singapore/Methodology)
[Kill Switch for Probiotics](https://2017.igem.org/Team%3ANUS_Singapore/Model_P)
[Kill Switch for BeeT](https://2017.igem.org/Team%3ANUS_Singapore/Model_B)

Projects

[Description](https://2017.igem.org/Team%3ANUS_Singapore/Description)
[Interlab Study](https://2017.igem.org/Team%3ANUS_Singapore/InterLab)
[Parts](https://2017.igem.org/Team%3ANUS_Singapore/Parts)
[Experiment](https://2017.igem.org/Team%3ANUS_Singapore/Experiments)
[Documentation](https://2017.igem.org/Team%3ANUS_Singapore/Notebook)
[Improvement](https://2017.igem.org/Team%3ANUS_Singapore/Improvement)
[Demonstrate](https://2017.igem.org/Team%3ANUS_Singapore/Demonstrate)
[Safety](https://2017.igem.org/Team%3ANUS_Singapore/Safety)
[Future Works](https://2017.igem.org/Team%3ANUS_Singapore/Future%20Work)

[Home](https://2017.igem.org/Team%3ANUS_Singapore)

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# Results

Here you can describe the results of your project and your future plans.

##### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

##### You should also describe what your results mean:

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember all measurement and characterization data must be on part pages in the Registry.
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

##### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

##### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)

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