iGEM 2019 — Team NUS_Singapore

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# Team:NUS Singapore/Notebook

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PROJECT

[Description](https://2019.igem.org/Team%3ANUS_Singapore/Description)
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PARTS

[Parts Overview](https://2019.igem.org/Team%3ANUS_Singapore/Parts)
[Basic Parts](https://2019.igem.org/Team%3ANUS_Singapore/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3ANUS_Singapore/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3ANUS_Singapore/Part_Collection)

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HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3ANUS_Singapore/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ANUS_Singapore/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3ANUS_Singapore/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3ANUS_Singapore/Hardware)
[Measurement](https://2019.igem.org/Team%3ANUS_Singapore/Measurement)
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[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=NUS_Singapore)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


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# Team:NUS Singapore/Experiments

NUS iGEM 2019

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[MEASUREMENT](https://2019.igem.org/Team%3ANUS_Singapore/Measurement)
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TEAM

[TEAM](https://2019.igem.org/Team%3ANUS_Singapore/Team)
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PROJECT

[DESCRIPTION](https://2019.igem.org/Team%3ANUS_Singapore/Description)
[DESIGN](https://2019.igem.org/Team%3ANUS_Singapore/Design)
[MEASUREMENT](https://2019.igem.org/Team%3ANUS_Singapore/Measurement)
[EXPERIMENTS](https://2019.igem.org/Team%3ANUS_Singapore/Experiments)
[RESULTS](https://2019.igem.org/Team%3ANUS_Singapore/Results)
[DEMONSTRATE](https://2019.igem.org/Team%3ANUS_Singapore/Demonstrate)
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[SAFETY](https://2019.igem.org/Team%3ANUS_Singapore/Safety)

HUMAN

[INTEGRATED HUMAN PRACTICES](https://2019.igem.org/Team%3ANUS_Singapore/Human_Practices)
[EDUCATION & ENGAGEMENT](https://2019.igem.org/Team%3ANUS_Singapore/Public_Engagement)

[ENTREPRENEURSHIP](https://2019.igem.org/Team%3ANUS_Singapore/Entrepreneurship)

[AWARDS](https://2019.igem.org/Team%3ANUS_Singapore/Awards)

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Lab notebooks

Toxin-antitoxin notebook

Click [here](https://static.igem.org/mediawiki/2019/9/92/T--NUS_Singapore--Experiments_Notebook_ToxinAntitoxin.pdf) to download our Toxin-antitoxin notebook.

SgrS notebook

Click [here](https://static.igem.org/mediawiki/2019/e/e0/T--NUS_Singapore--Experiments_Notebook_GlucoseTransporter.pdf) to download our SgrS notebook.

Plasmid retention and Biocontainment notebook

Click [here](https://static.igem.org/mediawiki/2019/6/6d/T--NUS_Singapore--Experiments_Notebook_PlasmidRetention_and_Biocontainment.pdf) to download our Plasmid retention and biocontainment notebook.

Improved part notebook

Click [here](https://static.igem.org/mediawiki/2019/7/73/T--NUS_Singapore--Experiments_Notebook_BlueLightPromoter.pdf) to download our Improved part notebook.

Protocols

As part of our team's collaborative efforts with the scientific community, we have uploaded a collection of our wet lab protocols on protocols.io.

LB media preparation
M9 media preparation
LB agar preparation
Antibiotics stock preparation (kanamycin, chloramphenicol, ampicillin)
Chemical preparation (IPTG, AHL, arabinose)
Chemically competent cell preparation
Q5 polymerase PCR cloning
DNA gel electrophoresis
Gel extraction

Hi-Fi Gibson assembly
Chemically competent cell transformation
Promega Glucose-Glo assay
Promega BacTiter-Glo assay
Colony Formation Unit (CFU)
Microplate reader workflow

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# Team:NUS Singapore/Results

NUS iGEM 2019

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[MEASUREMENT](https://2019.igem.org/Team%3ANUS_Singapore/Measurement)
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[DEMONSTRATE](https://2019.igem.org/Team%3ANUS_Singapore/Demonstrate)
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[MODEL](https://2019.igem.org/Team%3ANUS_Singapore/Model)
[SOFTWARE](https://2019.igem.org/Team%3ANUS_Singapore/Software)
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[SAFETY](https://2019.igem.org/Team%3ANUS_Singapore/Safety)
[INTEGRATED HUMAN PRACTICES](https://2019.igem.org/Team%3ANUS_Singapore/Human_Practices)
[EDUCATION & ENGAGEMENT](https://2019.igem.org/Team%3ANUS_Singapore/Public_Engagement)
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[AWARDS](https://2019.igem.org/Team%3ANUS_Singapore/Awards)

TEAM

[TEAM](https://2019.igem.org/Team%3ANUS_Singapore/Team)
[COLLABORATIONS](https://2019.igem.org/Team%3ANUS_Singapore/Collaborations)
[SPONSORS](https://2019.igem.org/Team%3ANUS_Singapore/Sponsors)

[DBTL](https://2019.igem.org/Team%3ANUS_Singapore/DBTL)

PROJECT

[DESCRIPTION](https://2019.igem.org/Team%3ANUS_Singapore/Description)
[DESIGN](https://2019.igem.org/Team%3ANUS_Singapore/Design)
[MEASUREMENT](https://2019.igem.org/Team%3ANUS_Singapore/Measurement)
[EXPERIMENTS](https://2019.igem.org/Team%3ANUS_Singapore/Experiments)
[RESULTS](https://2019.igem.org/Team%3ANUS_Singapore/Results)
[DEMONSTRATE](https://2019.igem.org/Team%3ANUS_Singapore/Demonstrate)
[ATTRIBUTIONS](https://2019.igem.org/Team%3ANUS_Singapore/Attributions)
[MODEL](https://2019.igem.org/Team%3ANUS_Singapore/Model)

[SOFTWARE](https://2019.igem.org/Team%3ANUS_Singapore/Software)

[PARTS](https://2019.igem.org/Team%3ANUS_Singapore/Parts)

[SAFETY](https://2019.igem.org/Team%3ANUS_Singapore/Safety)

HUMAN

[INTEGRATED HUMAN PRACTICES](https://2019.igem.org/Team%3ANUS_Singapore/Human_Practices)
[EDUCATION & ENGAGEMENT](https://2019.igem.org/Team%3ANUS_Singapore/Public_Engagement)

[ENTREPRENEURSHIP](https://2019.igem.org/Team%3ANUS_Singapore/Entrepreneurship)

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index

---

[Overview](#Overview)
[Growth Switch](#GrowthSwitch)
[Construction](#GrowthSwitch_construction)
[Characterization](#GrowthSwitch_characterization)
[Demonstration](#GrowthSwitch_demonstration)
[Blue-Light Control System](#GrowthSwitch_light_optimization)
[Demonstration of Blue-Light Control System](#GrowthSwitch_light_demonstration)
[Future directions](#GrowthSwitch_future_directions)
[Growth Knob](#GrowthKnob)
[Construction](#GrowthKnob_construction)
[Characterization](#GrowthKnob_characterization)
[Demonstration](#GrowthKnob_demonstration)
[Biocontainment](#Biocontainment)
[Construction](#Biocontainment_construction)
[Future Directions](#Biocontainment_future_directions)
[References](#References)

Overview

Until today, the ability for cells to stay viable for a long time while still capable of producing sufficient amounts of protein **remains a challenge** in many cell-based applications. This fundamental limitation prompted us to look **beyond existing preservation strategies** such as lyophilization and cryopreservation to find a more **sustainable and efficient solution** that can be used ***in situ***.
Here, we have developed a platform technology, known as **E.co LIVE**, to provide scientists with the option of adopting either a **growth switch** or a **growth knob** to control the growth and protein production profiles of cells **according to their needs**. We are pleased to share that our system has the ability to **regulate growth and protein production**, thereby prolonging the **functional lifespan** of *E. coli*.

GROWTH SWITCH

We have developed two growth switches using the toxin-antitoxin systems (i.e., HicA-HicB and RES-Xre) described in the **[description page](https://2019.igem.org/Team%3ANUS_Singapore/Description)** respectively to control growth and regulate protein production in *E. coli*.

Construction of growth switch

To construct our toxin and antitoxin plasmids, we employed the Gibson Assembly method. Appropriate primers were designed to amplify desired backbone and insert sequences, and the Gibson Assembly method was used to assemble those PCR products. The resulting Gibson products were subsequently transformed into *10-beta* competent *E. coli* cells.
Our bacteriostatic toxin and antitoxin genes were cloned into separate plasmids under the control of IPTG and arabinose-inducible promoters respectively (Fig. 1). Additionally, we incorporated a constitutive GFP cassette into the toxin plasmid to better understand the effect of toxin and antitoxin on protein production.
![](https://static.igem.org/mediawiki/2019/thumb/b/b8/T--NUS_Singapore--Design_Fig1.jpg/300px-T--NUS_Singapore--Design_Fig1.jpg)

*Fig. 1: Genetic circuits for (a) HicA-HicB and (b) RES-Xre systems in MG1655.*

To complete the growth switch, we co-transformed both toxin-GFP plasmid and antitoxin plasmid into *MG1655 knock-out HicA-HicB* strain (for HicA-HicB toxin-antitoxin system) and native *MG1655* strain (for RES-Xre toxin-antitoxin system).

Characterization of growth switch

1) Growth switch - “OFF button”

To test whether the growth switch is capable of arresting cell growth and act as an OFF button, we induced the cells containing our growth switch with IPTG after the cells were incubated in fresh LB media for an hour. Within an hour after the addition of IPTG, we saw a plateau in the growth curve of both the growth-arrested strains (Fig. 2a & 2b). The HicA-HicB system and RES-Xre system showed a significant growth arrested, even up to 12 hour (Fig. 2c & 2d).
![](https://static.igem.org/mediawiki/2019/thumb/5/56/T--NUS_Singapore--Results_Fig2.jpg/238px-T--NUS_Singapore--Results_Fig2.jpg.png)

*Fig. 2: Growth curve showing growth arrest in cells induced with (a) HicA toxin gene and (b) RES toxin gene expression after the addition of IPTG at 1h. (c) The extent of growth arrest by HicA toxin was found to be significant as shown by the difference in maximum OD600 and initial OD600 between HicA-mediated growth-arrested cells and normal-growing control cells. (d) Similarly, the extent of growth arrest by RES toxin was also significant.*

However, there was an unexpected resumption of growth in cells containing RES-Xre plasmids at approximately 10 hours, which was not observed in cells containing HicA-HicB plasmids. Previously, Skjerning et al. performed RES characterization for 6h, with no resumption of growth observed (Skjerning, Senissar, Winther, Gerdes, & Brodersen, 2019). We wondered if this auto-resumption of growth was simply not captured in the aforementioned work due to the shorter characterization duration. Hence, we proceeded to characterize the exact plasmid used in their work for 20h and saw a similar auto-resumption in growth despite the absence of Xre expression (Fig. 3). This confirmed that there is auto-resumption for the RES system. As the RES toxin functions to degrade cellular NAD+, it may be due to the existence of an inbuilt feedback system in the cells which helped to cope with the declining level of NAD+, thereby resulting in an auto-resumption of growth. Due to time constraints, we were unable to further validate this phenomenon. Nevertheless, quantifying intracellular NAD+ at different time points of the characterization can be done to validate this hypothesis for future work.
![](https://static.igem.org/mediawiki/2019/thumb/7/73/T--NUS_Singapore--Results_Fig3.jpg/240px-T--NUS_Singapore--Results_Fig3.jpg)

*Fig. 3: Growth curve of E. coli cells transformed with Skierning et al. RES plasmid, showing a resumption in growth, albeit much later (14 hour).*

2) Growth switch - “ON button”

To test whether our growth switch is capable of restoring cell growth and act as an 'ON' button, we induced the expression of antitoxin genes two and a half hours after the induction of toxin expression. As expected, the growth-arrested cells induced with antitoxin expression demonstrated a resumption in growth (Fig. 4a & 4b). Both HicA-HicB and RES-Xre systems showed a significant growth resumption (Fig. 4c & 4d). As for the cells containing RES-Xre plasmid, we observed an earlier resumption (at 5 hours) when arabinose was added to the induced antitoxin expression as compared to the auto-resumption which took place much later (at 9 hours) (Fig. 4b).
![](https://static.igem.org/mediawiki/2019/thumb/0/06/T--NUS_Singapore--Results_Fig4.jpg/244px-T--NUS_Singapore--Results_Fig4.jpg.png)

*Fig. 4: Growth curve of growth-arrested cells displaying resumption in growth after the induction of (a) HicB antitoxin and (b) Xre antitoxin gene expression with 0.0133M arabinose at 3.5h. (c) The extent of growth resumption by HicB antitoxin was found to be significant as shown by the difference in maximum OD600 and initial OD600 between growth-arrested cells and HicB-mediated growth-resumed cells. (d) Similarly, the extent of growth resumption mediated by Xre antitoxin was also significant.*

To prove that the toxins trigger growth arrest without killing the cells, we performed a Colony Forming Unit (CFU) assay before and after the induction of antitoxin expression in growth-arrested cells. The cells cultured in tubes had their growth arrested for 10 days prior to the CFU assay. We hypothesized that if the HicA toxin is bacteriostatic, expression of the HicB antitoxin should restore cell growth and allow cell division to take place. Hence, the number of colonies formed after the induction of antitoxin expression should be greater than that in growth-arrested cells without antitoxin expression. Consistent with our hypothesis, the results show that across all three dilutions, there were more colonies formed when growth-arrested cells were induced with antitoxin expression (Fig. 5). Taken together, the results demonstrated that the growth-arrested cells were able to remain viable even after 10 days of growth arrest, retaining the ability to grow and divide after antitoxin expression was induced.
![](https://static.igem.org/mediawiki/2019/thumb/b/b4/T--NUS_Singapore--Results_Fig5.jpg/320px-T--NUS_Singapore--Results_Fig5.jpg.png)

*Fig. 5: Growth-arrested cells maintained with 1mM IPTG for 10 days demonstrated the ability to restore growth and divide after the induction of HicB antitoxin expression as shown by (a) CFU plating and (b) CFU quantification at a 106 dilution factor.*

3) Growth switch to regulate protein production

After demonstrating HicA-HicB and RES-Xre ability to control cell growth, we proceeded to study the effect of our growth switch on protein production. This is so as protein production is usually coupled to growth (Sambrook & Russell, 2001). As such, we hypothesized that our growth switch can also function to regulate protein production. By measuring the level of GFP constitutively produced overtime, we observed growth-arrested cells displaying a suppressed level and lowered rate of GFP production compared to normal-growing control cells over a period of 12h (Fig. 6b). Statistical analysis was performed using nine experimental samples and the level of protein production was found to be significantly lower in growth-arrested cells compared to normal-growing cells (Fig. 6d). This suggests that growth arrest facilitated by our growth switch has the potential to act as an 'OFF' button to inhibit protein production.
Of note that RES-Xre system previously showed an incomplete growth arrest after the induction of RES toxin expression (Fig. 2b). Hence, we decided to only focus on the demonstration of HicA-HicB-mediated growth switch for downstream work.
![](https://static.igem.org/mediawiki/2019/thumb/c/c0/T--NUS_Singapore--Results_Fig6.jpg/247px-T--NUS_Singapore--Results_Fig6.jpg.png)

*Fig. 6: (a) Growth curve of growth-arrested cells induced with HicA toxin expression at 1h showing a reduced (b) total GFP and (c) average GFP production rate, demonstrating that the inhibition of cell growth inhibited its rate of protein production. (d) The level of protein production (maximum - initial) in HicA-mediated growth-arrested cells were found to be significantly lower compared to normal-growing control cells.*

With that being shown, we speculate that the induction of HicB antitoxin gene expression would likewise trigger our growth switch to restore GFP production rate. Indeed, cells showing growth resumption displayed a higher protein production rate in comparison with growth-arrested cells that were uninduced with antitoxin expression (Fig. 7b). Similarly, statistical analysis was performed using nine experimental samples and the level of protein production was found to be significantly higher in growth-resumed cells compared to growth-arrested cells (Fig. 7d). This demonstrated that our growth switch has the ability to restore protein production in previously growth-arrested cells, via the induction of antitoxin expression.
![](https://static.igem.org/mediawiki/2019/thumb/5/57/T--NUS_Singapore--Results_Fig7.jpg/247px-T--NUS_Singapore--Results_Fig7.jpg.png)

*Fig. 7: (a) Growth curve of growth-arrested cells induced with HicB antitoxin expression at 3.5h showing an increase in (b) total GFP and (c) average GFP production rate, demonstrating that the resumption of cell growth restored their protein production rate. (d) The level of protein production (maximum - initial) in HicB-mediated growth-resumed cells were found to be significantly higher compared to growth-arrested cells.*

Taken together, we are excited to show that our growth switch has the ability to control cell growth and regulate protein production, providing scientists with a tool to manipulate cells for fundamental and application-based studies.

4) Model prediction of growth switch activity

We understand that our growth switch was only tested with a single inducer concentration to trigger toxin and antitoxin expression, and we are cognizant that exploring different inducer concentrations could provide us with different growth profiles. Being inexperienced with the concentrations of inducers we should be adding in order to observe different growth and protein production profiles, our modelling team stepped in and constructed a reliable **[model](https://2019.igem.org/Team%3ANUS_Singapore/Model)** for HicA-HicB system, shown to nicely fit our experimental data.
Shown below are the few inducer concentrations ran by the wet lab to generate experimental data for the modelling team to construct a model of the system. We show that with an increasing concentration of arabinose explored, the rate of growth resumption increased accordingly, confirming that varying inducer concentrations alter the growth profile (Fig. 8)
![](https://static.igem.org/mediawiki/2019/thumb/f/f6/T--NUS_Singapore--Results_Fig8.jpg/240px-T--NUS_Singapore--Results_Fig8.jpg)

*Fig. 8: Growth curve of cells treated with fixed concentration of IPTG and varying concentrations of arabinose at 1h and 3.5h respectively to generate experimental data for model construction.*

Additionally, our growth switch should ideally be **(1)** achieving a maximum growth arrest upon induction of toxin expression, **(2)** showing a faster growth resumption upon the induction of antitoxin expression and **(3)** using the lowest possible inducer concentration to achieve (1) and (2). To find out the optimal inducer concentrations that could fulfil requirements (1) to (3), the modelling team came up with a **[surface plot](https://2019.igem.org/Team%3ANUS_Singapore/Model#Sensitivity)** (in the sensitivity analysis, growth switch section) which was useful at informing the wet lab team on the inducer concentrations they should be using or avoiding. By coupling a model prediction to our growth switch, we are able to provide users with predictions of growth profiles when parameters such as inducer concentration and induction time are varied. Check out our **[software tool](https://2019.igem.org/Team%3ANUS_Singapore/Software)** to find out more!

Demonstration of growth switch

After we have shown that our growth switch is able to work as intended to control growth and regulate protein production, we aim to couple our growth switch to relevant applications (such as bioluminescence production and cell-based AHL biosensors) so as to evaluate its ability to prolong the functional lifespan of these engineered cells. We identified the two applications for demonstration based on our findings from our human practice work, integrating their feedback and demand with our experimental direction.
As cellular resources are largely directed for growth, we wondered if intentional growth arrest could possibly conserve the pool of cellular resources originally directed for growth and only be utilized for protein production on demand at a later timepoint. This formed the basis of our goal in utilizing our HicA-HicB-mediated growth switch to prolong the functional lifespan of cells, allowing them to produce proteins even after a period of inactivity. In testing this hypothesis, we would also like to highlight that most of the demonstration work was performed using inducer concentrations which were recommended by our **[model](https://2019.igem.org/Team%3ANUS_Singapore/Model)**.

1) Inducible luminescence production

With an increasing demand for sustainable products in our daily lives, bioluminescent lighting is sought after as one of the alternatives to light up our surroundings, especially in rural areas. After interviewing **[Mr Prashant Mainali and the Universitas Indonesia 2019 iGEM team](https://2019.igem.org/Team%3ANUS_Singapore/Human_Practices)**, we learnt that scarce lighting remains a problem in some rural areas of Nepal and Indonesia. As such, we wondered if our growth switch could prolong the functional lifespan of luminescence-producing cells, keeping them viable until light is needed. Here, we demonstrate a prolonged production of luminescence in our growth-arrested cells in comparison with the normal-growing control cells. Replacing the aforementioned GFP cassette with a LuxCDABE operon under the control of arabinose-inducible promoter, we induced the cells with arabinose on day 5, day 10 and day 15 to trigger the production of luminescence (Fig. 9).
![](https://static.igem.org/mediawiki/2019/thumb/6/60/T--NUS_Singapore--Results_Fig9.jpg/320px-T--NUS_Singapore--Results_Fig9.jpg)

*Fig. 9: Genetic circuit of HicA-HicB toxin-antitoxin modules and LuxCDABE operon in MG1655.*

We show that on all three days, triggering of growth resumption and protein production resulted in growth-arrested cells producing a higher level of luminescence compared to normal-growing control cells (Fig. 10 & 11). Growth-arrested cells possess a lower starting OD600 value compared to normal-growing control cells due to the active inhibition of growth in the former (Fig. 10a - 10c).
![](https://static.igem.org/mediawiki/2019/thumb/e/eb/T--NUS_Singapore--Results_Fig10.jpg/268px-T--NUS_Singapore--Results_Fig10.jpg.png)

*Fig. 10: HicA-HicB-mediated growth switch generating a prolonged functional lifespan in luminescence-producing cells. (a), (d), (g) Growth curves showing growth-arrested cells with a lower starting OD600 indicative of growth inhibition across different days. (b), (e), (h) Total luminescence curves consistently showing a higher level of luminescence production upon in growth-arrested cells induced with HicA toxin expression, compared to normal-growing control cells without toxin expression. (c), (f), (i) Averaged luminescence curves showing a similar trend - growth-arrested cells producing higher level of luminescence per OD600 than normal-growing control cells.*

![](https://static.igem.org/mediawiki/2019/thumb/a/a0/T--NUS_Singapore--Results_Fig11.jpg/282px-T--NUS_Singapore--Results_Fig11.jpg.png)

*Fig. 11: Photo of normal-growing control cells and luminescent growth-arrested and revived cells taken on day 5.*

Taken together, this demonstration suggests the potential of our growth switch in implementing a more sustainable and controllable production of bioluminescence. As part of our human practice, we interviewed **[Ms Teresa van Dongen, a bioluminescent lamp designer](https://2019.igem.org/Team%3ANUS_Singapore/Human_Practices)**, and learnt that the inability to turn the bioluminescent lamp 'OFF' is one of its shortcomings. With our growth switch, we addressed this problem by allowing bioluminescence production to be turned 'ON' and 'OFF' at will, providing existing bioluminescence lighting with an enhanced function of acting much like an actual torchlight.

2) Cell-based AHL biosensor

As part of our human practice, we spoke to **[Ms Emily Hicks, the president of FREDsense technologies](https://2019.igem.org/Team%3ANUS_Singapore/Human_Practices)**, and understood that the need for a cold chain to keep their biosensor cells viable is a major bottleneck in their current operations. This led us to conceive of using our systems to enhance the performance of biosensors. To demonstrate the versatility of our growth switch in other applications, we coupled our growth switch to a cell-based diagnostic biosensor used for the detection of quorum sensing molecule (AHL) specific to pathogenic *Pseudomonas aeruginosa*. Through this demonstration, we aim to demonstrate the potential of prolonging cellular functional lifespan *in situ*. We first constructed the plasmids as shown in Fig. 12 and co-transformed both plasmids into *MG1655 E. coli*. Using the exact same set-up as the luminescence demonstration, we have growth-arrested cells and normal-growing control cells which differed in terms of *HicA* toxin expression. However, in this demonstration, a chemical AHL inducer was used to trigger *HicB* and *RFP* expression simultaneously instead of arabinose (Fig. 13).
![](https://static.igem.org/mediawiki/2019/thumb/9/95/T--NUS_Singapore--Results_Fig12.jpg/320px-T--NUS_Singapore--Results_Fig12.jpg)

*Fig. 12: Genetic circuit of HicA-HicB toxin antitoxin module incorporated into a biosensor system for AHL detection.*

![](https://static.igem.org/mediawiki/2019/thumb/f/fe/T--NUS_Singapore--Results_Fig13.jpg/245px-T--NUS_Singapore--Results_Fig13.jpg)

*Fig. 13: Pictorial diagram of cell-based AHL biosensor genetic circuitry. Growth-arrested cells were maintained with IPTG every three days to keep the cells dormant, reducing their rate of LasR production. Upon the presence of AHL, the basal level of LasR proteins produced would then activate LasI promoter to ‘wake’ the cells and produce reporter protein, RFP. On the other hand, normal-growing cells would be growing and constitutively producing LasR until AHL is detected to produce RFP.*

Here, we show that on day 11 the addition of AHL induced growth-arrested cells to produce a higher level of RFP compared to normal-growing control cells (Fig. 14b - 14c). Growth-arrested cells similarly possess a lower starting OD600 value compared to normal-growing control cells due to active growth inhibition in the former (Fig. 14a).
![](https://static.igem.org/mediawiki/2019/thumb/f/f9/T--NUS_Singapore--Results_Fig14.jpg/320px-T--NUS_Singapore--Results_Fig14.jpg.png)

*Fig. 14: HicA-HicB-mediated growth switch displaying a prolonged functional lifespan in cell-based AHL biosensor cells on day 11. (a) Growth curve of growth-arrested cells shows a lower starting OD600 compared to normal-growing control cells, which was indicative of growth inhibition up till day 11. (b) Total fluorescence curve shows growth-arrested cells producing more RFP protein than normal-growing cells, which was clearly supported by (c) average fluorescence curve whereby each cell produced on average a two-fold higher level of RFP protein.*

As part of our human practice, we spoke to **[Dr Karen Polizzi from Imperial College](https://2019.igem.org/Team%3ANUS_Singapore/Human_Practices)** and learnt that cell-based biosensors do not necessarily have to express high amounts of protein for detection. Hence, we speculate that the amount of RFP produced by growth-arrested cells (RFU= 6,000 - 10,000) on day 11 to be sufficient in biosensor applications, as long as this value exceeds the minimum detection threshold required for the application. While further work has to be done to optimize this system, taken together, we foresee the usefulness of our growth switch to be extremely relevant for *in situ* functions such as environmental and therapeutic monitoring. We thus propose E.co LIVE as a novel method to prolong the functional lifespan of engineered cells at 37°C, keeping them viable and functional *in situ* and removing reliance on a cold chain.

3) ATP Hypothesis

As mentioned previously, we hypothesize that HicA-mediated translational inhibition results in the accumulation of cellular reserves (e.g ATP) in growth-arrested cells, thereby allowing them to produce protein even after 10 days. This hypothesis is supported by Lobritz *et al.*, with their metabolomic data detailing a higher level of ATP in cells treated with the translation inhibitor chloramphenicol (Lobritz et al., 2015).
To validate this hypothesis, we measured the level of ATP present in normal-growing cells and growth-arrested cells on day 5. For both bioluminescence and biosensing demonstrations, we observed a higher level of ATP in growth-arrested cells as compared to normal-growing cells (Fig. 15). Statistical analysis was performed with four experimental samples for bioluminescence demonstration and the results were found to be significant. This preliminary observation is consistent with the observation in Lobritz *et al.*’s work, although further investigation is needed to clarify the possible effect of accumulated ATP on protein production.
![](https://static.igem.org/mediawiki/2019/thumb/7/75/T--NUS_Singapore--Results_Fig15real.jpg/320px-T--NUS_Singapore--Results_Fig15real.jpg.png)

*Fig. 15: (a) Total ATP level between growth-arrested cells and normal-growing cells in bioluminescence demonstration. Statistical analysis performed using four experimental samples revealed a significant difference in the level of ATP between both growth-arrested cells and normal-growing cells, suggesting that the inhibition of translation results in reduced ATP usage. (b) Similarly, total ATP level was found to be higher in growth-arrested cells compared to normal-growing cells for AHL biosensing demonstration. Statistical analysis was not performed due to insufficient experimental samples.*

Optimization of a blue-light controllable system as alternative input for the growth switch

Apart from regulating toxin and antitoxin expression using chemical inducible systems, we also explored other inducible systems to demonstrate the modularity and flexibility of our growth switch. Inspired by last year’s NUS iGEM team which tapped on the use of blue-light repressible promoter (BBa\_K2819103) to produce sustainable dyes, we decided to use the same promoter as a demonstration.
We first worked on improving the blue-light repressible promoter. The blue-light repressible promoter previously characterized by last year’s team contained a 11bp spacer between the promoter and ribosome-binding site (RBS). To further optimize the promoter activity, we attempted to remove and increase the length of spacer. To characterize the promoter, we used the same reporter protein from last year's iGEM team. To our delight, we found that the blue-light repressible promoter with 30bp spacer displayed an approximately 6-fold higher level of fluorescence production than the initial promoter with 11bp spacer (Fig. 16d). Meanwhile, the promoter without any spacer did not seem to produce any fluorescence at all. (This contributed to the **[new improved part](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3198005)**!).
![](https://static.igem.org/mediawiki/2019/thumb/2/26/T--NUS_Singapore--Results_Fig15.jpg/164px-T--NUS_Singapore--Results_Fig15.jpg.png)

*Fig. 16: Comparison of promoter activity over 8h. Growth curve of cells with varying spacer length (a) in the presence of blue light and (b) in the dark was plotted. Looking at the total RFP graph (c) in the presence of blue light and (d) in the dark, as well as the average RFP graph (e) in the presence of blue light and (f) in the dark, we clearly see that the blue-light repressible promoter with 30bp spacer consistently produced higher level of protein despite having a relatively higher OD600 than the other two samples, evidently shown by the average fluorescence curve.*

Demonstration of blue-light system as alternative input for growth switch

With an improved blue-light repressible promoter, we placed this promoter in front of *HicB* gene to control the expression of antitoxin. Meanwhile, the *HicA* gene is still under the control of IPTG-inducible promoter (Fig. 17). In this set-up, the presence of blue light results in the repression of *HicB*, while the removal of blue light activates the promoter for gene expression. Thus, we hypothesize that upon induction of toxin production and in the presence of blue-light, growth arrest will occur due to lack of antitoxin production. Check out our **[description page](https://2019.igem.org/Team%3ANUS_Singapore/Description)** to find out how the blue-light repressible promoter works!
![](https://static.igem.org/mediawiki/2019/thumb/b/bd/T--NUS_Singapore--Results_Fig16.jpg/291px-T--NUS_Singapore--Results_Fig16.jpg)

*Fig. 17: Genetic circuit showing HicA toxin gene placed under IPTG-inducible promoter and HicB antitoxin gene placed under blue-light repressible promoter containing a 30bp spacer. Meanwhile, the *EL222* gene is constitutively expressed.*

Here, we are proud to show that our growth switch coupled to the improved blue-light repressible promoter retained its desired functionality in response to the presence of blue light. Using a custom-made blue light LED illuminator, we could clearly see that cells pre-treated with IPTG grown under blue light produced visibly lesser GFP than cells treated with IPTG but grown in the dark (control cell 1) and also untreated cells grown under blue light (control cell 2). This was consistent with our hypothesis (Fig. 18).
![](https://static.igem.org/mediawiki/2019/thumb/9/97/T--NUS_Singapore--Results_Fig17.jpg/320px-T--NUS_Singapore--Results_Fig17.jpg.png)

*Fig. 18: Microplate showing a visual representation of GFP production under a blue light illuminator for (a) control cells shone with blue light only, (b) test cells induced with IPTG and shone with blue light, (c) control cells induced with IPTG but kept in the dark. All three conditions were performed for 24h before visualization.*

To visualize for the recovery of GFP production, we left the plate to shake in the dark for 24h. Formerly growth-arrested cells were subsequently observed to produce comparable amounts of GFP when compared to both controls, indicating a recovery in protein production (Fig. 19).
![](https://static.igem.org/mediawiki/2019/thumb/2/28/T--NUS_Singapore--Results_Fig18.jpg/320px-T--NUS_Singapore--Results_Fig18.jpg.png)

*Fig. 19: Microplate showing visual representation of GFP production under a blue light illuminator for (a) control cells exposed to blue light only, (b) test cells induced with IPTG and exposed to blue light, (c) control cells induced with IPTG but kept in the dark. All three samples were kept in the dark for 24h before visualization.*

To further validate that the growth switch is capable of controlling growth and regulating protein production in response to blue light, we proceeded to characterize the cells using a microplate reader. After incubating the cells that were pre-treated with IPTG under blue light for the whole night, we measured cell growth and level of fluorescence production by the cells the next day. As expected, a plateau was observed at the beginning of the measurement, indicating that cell growth was indeed arrested from overnight treatment. Furthermore, the starting OD600 of cells treated with IPTG and exposed to blue light was relatively lower than the light-only control cells, which further supports the hypothesis that growth was arrested in the former cells. Over time, it appeared that absence of blue light in the microplate reader triggered the expression of antitoxin. resulting in growth resumption as shown in Fig. 20a.
![](https://static.igem.org/mediawiki/2019/thumb/2/26/T--NUS_Singapore--Results_Fig19.jpg/320px-T--NUS_Singapore--Results_Fig19.jpg.png)

*Fig. 20: Effect of blue-light controlled growth switch on (a) cell growth (b) total protein production and (c) average protein production when growth-arrested cells were kept in dark and measured for 12h.*

From the experiments conducted above, a positive trend between cell growth and protein production under the control of blue light was observed, indicating that the presence of blue light and IPTG induced growth arrest. As a result, a lower level of GFP was produced at the beginning of the measurement (Fig. 20b). Over time, we see that the level of GFP produced increased as the absence of blue light triggered the production of antitoxin, thereby ‘waking’ the cells up to produce more GFP. The validity of these results are tempered by the variance observed in the readings, necessitating further improvements in experimental designs despite the overall clear trend.
Taken together, we show that our growth switch developed to control cell growth and regulate protein production is functional even when it is partially controlled by a blue-light inducible system. As such, we envision the adoption of other inducible systems (e.g temperature inducible system) to also result in a functional growth switch with the ability to prolong cellular functional lifespan. Through this demonstration, we were able to showcase the flexibility and modularity of our growth switch, making it a broad and useful toolbox for scientists.

Future directions

Having demonstrated the capability of our HicA-HicB-mediated growth switch to prolong the functional lifespan of cells, we are interested in further optimizing the level and duration of protein production in these cells for our future work. We would also like to characterize other toxin-antitoxin genes to study their ability to control growth and regulate protein production, so as to further improve our growth switch.
As our current growth switch was only performed at 37°C to control cellular activity, we would also like to look into utilizing the growth switch at ambient room temperature. By doing so, we aim to ultimately bypass the need for a temperature-controlled supply chain to keep the cells viable and at the same time, functional in cell-based applications.

GROWTH KNOB

Aside from an 'ON/OFF' growth switch, we have also developed a growth knob which utilizes a small RNA, SgrS, to target the glucose uptake system - thereby regulating both the growth rate and protein production rate in a continuous manner akin to a tunable growth knob.

Construction of growth knob

To construct the SgrS system, we employed the Gibson Assembly method to clone SgrS fragment obtained from BBa\_K581005 into a compatible backbone plasmid containing a tetracycline-inducible promoter (Fig. 21). Afterwards, we transformed the Gibson products into *10-beta* cells during the cloning process and eventually transformed the purified plasmid into *MG1655* for characterization purposes.
![](https://static.igem.org/mediawiki/2019/thumb/c/c5/T--NUS_Singapore--Results_Fig20.jpg/320px-T--NUS_Singapore--Results_Fig20.jpg)

*Fig. 21: Genetic circuit of SgrS gene placed under the control of tetracycline-inducible promoter.*

Characterization of growth knob

Unlike the growth switch which toggles the growth between dormant and active state, our growth knob is developed to control the growth rate of cells and as a result, generate a more continuous growth and protein production profile.
With SgrS expression controlled by a tetracycline-inducible promoter, we explored a range of aTc inducer concentrations to study the effect of SgrS on cell growth. Since SgrS functions by binding to ptsG mRNA (encoding for one of the glucose transporter subunits) to prevent its translation, we hypothesize that an increasing concentration of aTc would likely result in greater extent of growth rate reduction in cells due to the corresponding increase in SgrS RNA resulting in further downregulation of ptsG expression. As a result, with a lower glucose uptake rate, cell growth is likely to be negatively affected since glucose is known to be preferentially metabolized during cell growth (Rosano & Ceccarelli, 2014).
We observed results that are consistent with our hypothesis. Increasing aTc concentration beyond 10nM results in a slower growth rate (Fig. 22a). Importantly, we also observed that an increasing concentration of aTc resulted in lower glucose consumption and a lowered growth rate throughout 18h of microplate readings (Fig. 22b).
![](https://static.igem.org/mediawiki/2019/thumb/7/78/T--NUS_Singapore--Results_Fig21.jpg/320px-T--NUS_Singapore--Results_Fig21.jpg.png)

*Fig. 22: (a) Growth curve of cells induced with different aTc concentrations showing a negative correlation between aTc concentration and growth rate of cells. (b) Looking at different aTc concentrations used during the characterization, higher aTc concentrations reflected a lower level of glucose consumption as well as a smaller increase in growth, compared to lower aTc concentrations.*

Taken together, these results support our hypothesis - that controlling cellular glucose uptake rate can effectively control cellular growth rate and produce different growth trends.

Demonstration of growth knob

As protein production is usually coupled to growth, we hypothesize that cells treated with higher aTc concentrations will likely produce less proteins since their growth rate is reduced. However, we should expect protein production in these cells to last a longer period of time due to the accumulation of unused glucose in the media.
To demonstrate that the growth knob can produce not just differential growth but also a spectrum of protein production trends, we co-transformed *MG1655* with SgrS plasmid and a simple plasmid containing an *RFP* gene placed under the control of an IPTG-inducible promoter. As expected, different extents of growth reduction mediated by different aTc concentrations produced protein production trends which are aligned to our hypothesis (Fig. 23). However, due to the slow degradation rate of the RFP protein, we were unable to show the entire protein production profile. Nevertheless, future work could include the use of degradation tags to study the duration of protein production in cells treated with varying aTc concentrations for our future work.
![](https://static.igem.org/mediawiki/2019/thumb/b/be/T--NUS_Singapore--Results_Fig22.jpg/320px-T--NUS_Singapore--Results_Fig22.jpg.png)

*Fig. 23: SgrS-mediated growth knob regulating protein production. (a) Growth curve of cells co-transformed with SgrS plasmid and inducible RFP plasmid showing a negative correlation between aTc concentration and growth rate of cells. (b) Total RFP graph revealed a spectrum of protein production profiles - increasing aTc concentrations showing a decreasing protein production rate and generating a gentler protein production profile as hypothesized. (c) Average RFP produced by cells treated with different aTc concentrations was plotted to support the analysis.*

Although a simple reporter gene was used to demonstrate the functionality of our growth knob, we envision this growth knob to be useful at fine-tuning growth and protein production in applications requiring different protein production profiles. For this growth knob, we have also constructed a model based on the experimental data generated by our wet lab team. To check out the different simulations we have run with this model click **[here](https://2019.igem.org/Team%3ANUS_Singapore/Model)**!

BIOCONTAINMENT CUM PLASMID RETENTION

To minimize the contribution of our system towards antibiotic resistance and yet retain the plasmid-of-interest in the cell, we came up with an unconventional concept for our final E.co LIVE system without having to rely on antibiotic use.

Construction of dual plasmid retention system

To construct our dual plasmid retention system, we employed the Gibson Assembly method to clone our IDT-synthesized toxin-antitoxin gBlock into compatible backbones, thereby generating two complementary plasmids (Fig. 24). In this system, *Hok-Sok* and *Txe-Axe* are two different toxin-antitoxin systems where the former gene encodes for toxin protein and the latter encodes its cognate antitoxin protein.
![](https://static.igem.org/mediawiki/2019/thumb/7/7a/T--NUS_Singapore--Results_Fig23.jpg/240px-T--NUS_Singapore--Results_Fig23.jpg)

*Fig. 24: Genetic circuits showing each plasmid containing an opposing pair of toxin and antitoxin to generate selection pressure for plasmid retention. The top plasmid contains Axe antitoxin from Txe-Axe toxin-antitoxin system, Hok toxin from Hok-Sok toxin-antitoxin system, RFP gene and chloramphenicol resistance gene. On the other hand, the bottom plasmid contains Txe toxin from Txe-Axe toxin-antitoxin system, Sok antitoxin from Hok-Sok toxin-antitoxin system, GFP gene and kanamycin resistance gene.*

After obtaining the Gibson products for both plasmids, we transformed them into *10-beta* cells for cloning and sent the extracted plasmids for sequencing. Interestingly, we discovered that one of the toxin pairs, more specifically *Hok*, contains a nonsense mutation encoding for a stop codon within the gene itself (Fig. 25). This renders the Hok-Sok toxin-antitoxin pair non-functional as the *Hok* gene has likely lost their ability to enforce selection pressure on the cells.
![](https://static.igem.org/mediawiki/2019/thumb/1/11/T--NUS_Singapore--Results_Fig24.jpg/320px-T--NUS_Singapore--Results_Fig24.jpg)

*Fig. 25: Sequencing results showing that the deletion and point mutation changed tyrosine (TAC) into a stop codon (UAA) at amino acid position 35.*

Nevertheless, we continued to transform both plasmids into *MG1655* and plated the cells on either chloramphenicol or kanamycin plates to test their functionality. For cells plated on the chloramphenicol plate, we observed most colonies to be red and non-fluorescing (Fig. 26a). This was expected because despite having a mutated *Hok* toxin gene, the chloramphenicol resistance gene and *RFP* gene present in the same plasmid are still functional, allowing the cells to grow and form red colonies. The reason why we expected fewer orange colonies on chloramphenicol plate, was because the presence of mutated *Hok* gene would have rendered the other plasmid containing the ‘antidote’ *Sok* and *GFP* unnecessary since it is no longer required to neutralize the Hok toxin, which is unable to impose a killing pressure on the cells.
On the other hand, we observed that most colonies were orange in color when cultured on a kanamycin plate (Fig. 26b). This was expected because sequencing results revealed the *Txe* toxin gene to be intact. As such, Txe toxins produced have to be neutralized by its cognate antitoxin Axe, which was transcribed and translated from the other plasmid. Hence, for cells to survive on a kanamycin plate, both plasmids have to be taken up by the cells, thus causing both GFP and RFP to be concurrently produced and resulting in an orange color formed. Altogether, this demonstrates that despite having only one pair of functional toxin-antitoxin (Txe-Axe) present in separate plasmid, almost all of the transformed cells could still retain both plasmids as shown in Fig. 26b. More importantly, the results provide preliminary insight into the feasibility of our conceptual dual plasmid retention system.
![](https://static.igem.org/mediawiki/2019/thumb/d/dc/T--NUS_Singapore--Results_Fig25.jpg/320px-T--NUS_Singapore--Results_Fig25.jpg.png)

*Fig. 26: MG1655 transformed with both plasmids plated on (a) chloramphenicol and on (b) kanamycin, visualized with a blue light illuminator. Under the blue light illuminator with a wavelength ranging between 380nm to 500nm, GFP with the excitation wavelength of 485nm would be able to fluoresce while RFP with the excitation wavelength of 528nm would not be able to fluoresce. However, as the emission wavelength of GFP (528nm) was coincidentally the same as the excitation wavelength of RFP, cells containing both GFP and RFP would fluoresce to produce an orange fluorescence as shown in (b).*

Future directions

Due to time constraints, we were unable to correct the nonsense mutation and revert *Hok* back into a functional form. Future work would entail re-cloning the plasmid and sending it for sequencing to verify if the mutation happens at the same position again. If it does, we hypothesize that the cells are likely to have a strong selection against the expression of *Hok* gene, resulting in deliberate mutationd to inactivate its function. Explorig other bactericidal toxin-antitoxin systems and study their ability to retain plasmids and minimize leaking of genes into the environment would then be the next course of action.
Further investigations into this system would include investigating the killing efficiency of a single plasmid (without its cognate antitoxin) compared to cells with both plasmids, as well as the extent of plasmid retention for dual plasmid retention system in comparison with common antibiotic use.

REFERENCES

Lobritz, M. A., Belenky, P., Porter, C. B. M., Gutierrez, A., Yang, J. H., Schwarz, E. G., … Collins, J. J. (2015). Antibiotic efficacy is linked to bacterial cellular respiration. Proceedings of the National Academy of Sciences of the United States of America. https://doi.org/10.1073/pnas.1509743112
Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: Advances and challenges. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2014.00172
Sambrook, J., & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual, Third Edition. In Molecular Cloning: a laboratory a manual,.
Skjerning, R. B., Senissar, M., Winther, K. S., Gerdes, K., & Brodersen, D. E. (2019). The RES domain toxins of RES-Xre toxin-antitoxin modules induce cell stasis by degrading NAD +. Molecular Microbiology. https://doi.org/10.1111/mmi.14150

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