iGEM 2018 — Team Leiden

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Leiden/Notebook

Although the mobile version has the same content, this wiki is best viewed on larger screens

# Notebook

During the course of our labwork we have made sure to document all experiments and experimental conditions in our notebook. This has allowed us to improve on and learn from each of our experiments. To that end, we hope providing our full
notebook online will help others trying to recreate our system improve upon it.

pSB1C3 construct preparation

Chromoproteins

Composite part construction

Gram-negative promoter isolation

Gram-positive promoter isolation

Proof of concept - promoters

Proof of concept - system

[ProtocolsClick here to read the protocols we used during our experiments](https://2018.igem.org/Team%3ALeiden/Experiments)
[PartsClick here to view all the parts we created during our project](https://2018.igem.org/Team%3ALeiden/Parts)
[SafetyClick here to read how we ensured a safe and secure lab environment during our project](https://2018.igem.org/Team%3ALeiden/Safety)


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Leiden/Experiments

Although the mobile version has the same content, this wiki is best viewed on larger screens

# Protocols

On this page you can find a list of protocols we used and optimized during our research, each detailing how to carry out experiments the way we performed them. Simply click on a protocol to read its steps. For easy navigation, you can use
our
side menu to scroll to the protocol you are looking for.

Pouring Agar plates

1. Add LB-agar to dH2O in the prescribed concentration
2. Autoclave the obtained LB-agar solution
3. Let the solution cool down to 60°C and add antibiotics where necessary
4. Pour the LB-agar in empty Petri dishes until it covers the bottom
5. Leave the Petri dishes parially open (asceptically) until the agar is completely solidified (approximately 5 minutes)

Final concentration of antibiotics:* Ampicillin: 100 μg/mL
* Chloramphenicol: 25 μg/mL
* Kanamycin: 50 μg/mL
* Tetracyclin: 10 μg/mL

Competent *E. coli* (DH5α)

1. Grow *E. coli* (DH5α) in LB medium at 37°C overnight
2. Transfer a small fraction of the overnight culture into fresh LB and grow untill the culture reaches an OD600 of about 0.3-0.4
3. Pellet the cells at 4000 rpm for 10 minutes
4. Resuspend the cells in TSS buffer
5. Make aliquots of a desired volume and store at -80°C

**TSS buffer** is composed of 10% (w/v) polyethylene glycol, 5% (v/v) dimethyl sulfoxide (DMSO) and 50 mM Mg2+ set at pH 6.5.

Competent *Bacillus subtilis* (strain 168) and subsequent transformation

1. Make an overnight liquid culture of *B. subtilis*, grown at 37°C
2. Transfer the bacteria to SM1 medium (37°C) and incubate at 37°C until an OD600 of 0.5 to 0.7 is reached (4 to 5 hours)
3. Add an equal volume of SM2 medium (37°C) and incubate for another 90 minutes
4. Transfer the suspension to a microcentrifuge tube and add 1 μL of ligated construct solution
5. Incubate at 37°C for 30 minutes under shaking conditions (200 rpm)
6. Add 60 μL LB medium and incubate for another 30 minutes before plating on appropriate plates

**SM1 medium** is made with 15 mL ST-base, 150 μL Tryptophane solution (10 mg/mL), 37.5 μL MgSO4 (0.3 M), 300 μL 50x YECA and, 150 μL 50% glucose

**SM2 medium** is made with 15 mL ST-base, 150 μL Tryptophane solution (10 mg/mL), 150 μL MgSO4 (0.3 M), 150 μL 50x YECA, 150 μL 50% glucose and, 75 μL CaCl2

Genomic DNA isolation *E. coli*
Using GenElute™ Bacterial Genomic DNA Kit

1. Pellet an overnight culture of *E. coli* (5 minutes @ 13 000 rpm) and discard the supernatant
2. Resuspend the pellet thoroughly in 180 μL lysis solution and 20 μL RNase
3. Incubate this solution for 2 minutes at room temperature
4. Add 20 μL of Proteinase K solution and incubate another 30 minutes at 55°C
5. Add 200 μL of Lysis Solution C and vortex
6. After 10 minutes of incubation at 55°C, add 200 μL of ethanol (95-100%) to the lysate and mix thoroughly
7. Transfer the contents into a binding column and spin at ≥6500 ×g for 1 minute
8. Discard the eluate
9. Wash the colum with washing solution, spin down and discard the eluate
10. Repeat the washing step with the same washing solution, this time supplemented with ethanol
11. To elute the DNA, add 200 μL Elution solution and incubate for 5 minutes at room temperature
12. Collect the DNA by spinning down the sample at ≥6500 ×g for 1 minute

Plasmid DNA isolation *E. coli*
Using GenElute™ Plasmid miniprep Kit

1. Pellet an overnight culture of *E. coli* (5 minutes @ 13 000 rpm) and discard the supernatant
2. Resuspend the pellet in 200 μL Resuspension solution
3. Add 200 μL Lysis solution and invert gently to mix (**Do not vortex!**)
4. Incubate this solution for 5 minutes at room temperature
5. Add 350 μL of Neutralization solution and invert to mix
6. Pellet the debris (10 minutes @ max-speed)
7. Meanwhile, wash the binding column with 500 μL Column preparation solution (1 minute @ max-speed) and discard the eluate
8. Transfer the cleared lysate (supernatant from step 6) into a binding column and spin down at ≥12000 ×g for 1 minute
9. Discard the eluate
10. Wash the colum with 750 μL washing solution, spin down and discard the eluate
11. Spin the column 1 minute to dry the column and then transfer the column to a new collection tube
12. To elute the DNA, add 100 μL Elution solution and spin down the sample at max-speed for 1 minute

PCR on genomic DNA

1. Prepare the PCR reaction as is indicated below:

| Component | Volume (final concentration) |
| --- | --- |
| Taq polymerase buffer (10x) | 5 μL |
| Taq polymerase | 1 μL |
| dNTPs | 4 μL (200 μM total) |
| Forward primer | 0.5 μL (0.5 μL) |
| Reverse primer | 0.5 μL (0.5 μL) |
| DMSO | 1.25 μL |
| MilliQ | 36.75 μL |
| Total | 49 μL |

2. Add 1 μL of genomic DNA (approximately 200 ng) to the reaction mix
3. Put the sample(s) in a PCR machine and run the following protocol:

|  |  |
| --- | --- |
| Step 1 (1x) | 2 min @ 94°C |
| Step 2 (30x) | 30 sec @ 94°C  30 sec @ 50°C  30 sec @ 72°C |
| Step 3 (1x) | 5 min @ 72°C  hold @ 12°C |

4. Check the PCR products by performing gel electrophoresis

Tip: Don't forget to make a sacrifice to the PCR gods in order for it to succeed

Single BioBrick Assembly

1. Restrict the desired BioBrick part with EcoRI and PstI in a suitable buffer for 1 hour at 37°C

   Below you can find the composition of restriction solution that we used:

| Compound | Volume |
| --- | --- |
| Restriction enzymes | 1 μL each |
| Cutsmart buffer | 2 μL |
| BioBrick DNA | 5 μL |
| MilliQ | 11 μL |

2. Restrict the destination plasmid with EcoRI and PstI in a suitable buffer for 1 hour at 37°C

   Below you can find the composition of restriction solution that we used:

| Compound | Volume |
| --- | --- |
| Restriction enzymes | 1 μL each |
| Cutsmart buffer | 3.5 μL |
| Backbone plasmid DNA | 3 μL |
| MilliQ | 11.5 μL |

3. Deactivate the restriction enzymes at 80°C for 20 minutes
4. Ligate the cut BioBrick and destination plasmid with T4 ligase for 1 hour at room temperature

   Below you can find the composition of ligation solution that we used:

| Compound | Volume |
| --- | --- |
| BioBrick part | 12 μL |
| Plasmid backbone | 5 μL |
| T4 Ligase buffer | 2 μL |
| T4 Ligase | 1 μL |

5. Transform *E. coli* with the obtained mixture according to the transformation protocol

Dual BioBrick Assembly

1. Restrict the desired upstream BioBrick part with EcoRI and SpeI in a suitable buffer for 1 hour at 37°C

   Below you can find the composition of restriction solution that we used:

| Compound | Volume |
| --- | --- |
| Restriction enzymes | 1 μL each |
| Cutsmart buffer | 2 μL |
| BioBrick DNA | 5 μL |
| MilliQ | 11 μL |

2. In the same manner, restrict the desired downstream BioBrick part with XbaI and PstI in a suitable buffer for 1 hour at 37°C
3. Restrict the destination plasmid with EcoRI and PstI in a suitable buffer for 1 hour at 37°C

   Below you can find the composition of restriction solution that we used:

| Compound | Volume |
| --- | --- |
| Restriction enzymes | 1 μL each |
| Cutsmart buffer | 3.5 μL |
| Backbone plasmid DNA | 3 μL |
| MilliQ | 11.5 μL |

4. Deactivate the restriction enzymes at 80°C for 20 minutes
5. Ligate the cut BioBricks and destination plasmid with T4 ligase for 1 hour at room temperature

   Below you can find the composition of ligation solution that we used:

| Compound | Volume |
| --- | --- |
| BioBrick parts | 5 μL each |
| Plasmid backbone | 3 μL |
| T4 Ligase buffer | 2 μL |
| T4 Ligase | 1 μL |
| MilliQ | 4 μL |

6. Transform *E. coli* with the obtained mixture according to the transformation protocol

DNA purification *E. coli*: Quick and Dirty

1. Transfer a single colony of transformants into liquid LB medium with appropriate antibiotic and incubate overnight at 37°C
2. Pellet the cells (5 minutes @ 13 000 rpm) and discard the supernatant
3. Resuspend the cells in 100 μL STET buffer
4. Incubate the solution at room temperature for 5 minutes
5. Add 5 μL lysozyme solution (10 mg/mL)
6. Incubate 1 minute at 95 to 100°C
7. Centrifuge the sample (13 000 rpm for 10 minutes) and continue with the supernatant
8. Add 105 μL cold isopropanol (-20°C) and vortex slightly
9. Centrifuge the sample (13 000 rpm for 10 minutes) and discard the supernatant
10. Add 400 μL ethanol (70%) to the pellet and vortex shortly
11. Centrifuge the sample at 13 000 rpm for 5 minutes
12. Discard the supernatant and dry the pellet at 65°C for 10 minutes
13. Resuspend pellet in 50 μL TE buffer containing 1 μL RNase
14. Incubate at 65°C for 20 minutes and vortex
15. The obtained solution contains the desired DNA and can be used for gel electrophoresis to check whether a transformation was successfull

**STET buffer** is composed of 8% sucrose, 0.5% Triton X-100, 50 mM Tris-HCl and 50 mM EDTA (pH 8.0)

**TE buffer** is composed of 10 mM TRIS and 1 mM EDTA (pH 8.0)

DNA purification *B. subtilis*: Quick and Dirty

1. Grow an overnight culture of *B. subtilis*
2. Pellet the bacteria (5 minutes @ 13 000 rpm) and discard the supernatant
3. Resuspend the bacteria in 100 μL TEG buffer
4. Add lysozyme solution and incubate for 10 minutes at 37°C
5. Add NaOH/SDS solution and mix until it clarifies
6. Add NaAC (3 M) to precipitate the cell components
7. Centrifuge solution at 13 000 rpm for 7 minutes and continue with the supernatant
8. Add isopropanol (-20°C) and mix the solution
9. Pellet the sample (5 minutes @ 13 000 rpm) and discard the supernatant
10. Add 400 μL ethanol (70%)
11. Pellet the samples again and discard the supernatant
12. Dry the pellet for 10 minutes at 65°C
13. Resuspend the pellet in 50 μL TE buffer supplemented with 1 μL RNase and incubate at 65°C for 20 minutes

**TEG buffer** is composed of 25% (w/v) sucrose, 50 mM EDTA and, 50 mM Tris-HCl (pH 8.0)

**NaOH/SDS solution** is composed of 0.2 M NaOH and 1% (vol/vol) SDS in MilliQ

Measure DNA concentration (NanoDrop)

1. Initiate the dsDNA program of the NanoDrop
2. Clean the sensor and blank with 1 μL sterile MilliQ
3. Load 1 μL sample and measure the DNA concentration

Note: The A260/280 ratio should be larger than 1.8 and the A260/230 should be larger than 2.0

Gel electrophoresis

Note: This protocol uses Ethidium Bromide, which is a carcinogenic compound. Handle all infected materials with a glove.

1. Heat a 1% agarose TAE buffer solution to dissolve the agarose
2. Add Ethidium Bromide to a final concentration of 0.5 μL/mL and transfer the mixture to a gel electrophoresis mold
3. Allow the gel to solidify
4. Place the gel into an electrophoresis device filled with TAE buffer and load the desired samples and appropriate DNA ladder(s)
5. Run the gel at 100 V for 20 to 60 minutes
6. Transfer the gel to a gel imager and visualize the DNA

Transformation *E. coli*

1. Thaw competent *E. coli* cells on ice
2. Add 5 μL of ligated construct solution to roughly 50 μL cells and mix carefully by pipetting up and down
3. Incubate the sample on ice for 30 to 60 minutes
4. Incubation the sample at 42°C for 90 seconds (heat shock) before returning it to ice again
5. Keep cells on ice for 5 minutes
6. Add 1 mL LB medium to the sample and grow at 37°C for about an hour under shaking conditions (200 rpm)
7. Plate the obtained solution on LB agar plates with appropriate antibiotic(s) and incubate overnight

Long-term storage of bacteria

1. Grow an overnight culture of the desired bacterial strain at the appropriate conditions
2. Add twice the volume of 20% glycerol solution and mix thoroughly
3. Store the bacteria at -20°C to -80°C

Tip: Make sure to label the vials extensively to prevent mistakes later on

Preparation for flow cytometry

1. Make an overnight culture of the strain(s) to be tested
2. Dilute the bacteria 50 to 200 times in LB medium (aiming for an OD600 of 0.1 approx.)
3. Transfer 100 μL culture to each well of a U-shaped 96-well plate
4. Add 100 μL of stressor solution to each well
5. Incubate the plate in a microplate incubator shaker at 37°C, 600 rpm for 2 to 4 hours
   Keep bacteria on ice for the subsequent steps
6. Centrifuge the 96-well plate at 2000 ×g for 5 minutes
7. Discard the supernatant and resuspend the cells in ice-cold PBS buffer (-4°C)
8. Centrifuge again and repeat the washing step with PBS
9. Analyze samples by flow cytometry

Disk diffusion

1. Inoculate an indicator strain in LB medium and incubate at 37°C untill an OD600 of 0.1 is reached
2. Distribute the liquid culture uniformly over an Mueller-Hinton agar plate with a sterile cotton swab
3. Carefully place felt disks on the agar plate and pipette 35 μL of antibiotic-LB solution into the disk
4. Incubate at 37°C for 24-72 hours

Note: Do not place the petri dish upside down to prevent the disk from falling off

Overlay assay with *Streptomyces*

1. Prepare ISP 2 medium agar plates
2. Make 6 spots (evenly distributed across the plate) of 1 μL glycerol-stock of *Streptomyces* (e.g. *S. coelicolor* and *S. Griseus* in different dilutions)
3. Incubate the *Streptomyces* spots for 48 hours at 30°C
4. Meanwhile, grow an indicator strain at 37°C untill an OD600 between 0.4 and 0.6 is reached
5. Make a solution of 2% (v/v) indicator strain in soft agar
6. Pour approximately 5 to 10 mL of the soft agar mixture evently on top of the *Streptomyces* spots
7. Incubate overnight at 37°C

**ISP 2 medium agar plates** is composed of 0.4% (w/v) yeast extract, 1% (w/v) malt extract, 0.4% (w/v) dextrose and 2% (w/v) agar in distilled water

**Soft agar** is a 1% agarose in LB solution (half the agar concentration than LB agar)

Freeze drying *E. coli*

1. Make an overnight culture of the desired *E. coli* strain
2. Plate 100 μL of that culture on a petri dish with LB agar
3. Incubate overnight at 37°C
4. Add 4 mL of a 1% glycerol solution to the plates and scrape the bacteria loose
5. Prepare plastics tube for freeze drying by making a hole in the lid to prevent pressure build up in the freeze dryer
6. Transfer the obtained suspension to the plastic tube
7. Place the tubes in a freeze dryer container and let the freeze dryer run for at least 24 hours
8. Carefully remove the tubes and store the bacteria at room temperature

Confocal microscopy

1. Grow overnight culture of the desired strains
2. Dilute to an OD600 of 0.1
3. Add stressors to the desired concentration (if applicable)
4. Incubate further for 2 to 3 hours
5. Place a droplet of the culture on a microscopy slide and view under the microscope

Our confocal microscopy pictures were taken with a Zeiss LSM 5 Exciter-Axiolmager M1 confocal microscope with a Plan-Aprochromat objective (63x/1.4 Oil DIC) and Zeiss Zen 2009 software. For GFP measurements, cells were excited at 488 nm
and measured using an emission filter of 505-600 nm. *E. coli* cells with mCherry were excited at 575 nm and emission at 610 nm.

Lethality screening of natural compounds

1. Prepare stocks of soluble compounds in MilliQ. If using natural products, soak the compounds in milliQ for 24 hours at 60°C. The table below outlines the concentrations used of the products in our experiments.

   | Product | Concentration (g/mL) |
   | --- | --- |
   | Garlic (extract) | 1.10 |
   | Curcuma (extract) | 0.20 |
   | Onion (extract) | 1.00 |
   | Honey | 0.80 |
   | Red pepper (extract) | 1.47 |
   | Ginger (extract) | 0.31 |
   | Cinnamon (extract) | 0.26 |
   | Caffeine | 0.04 |
   | Oregano (extract) | 0.20 |
   | Thyme (extract) | 0.20 |
   | Liquorice (extract) | 0.21 |
   | Ginger pre-packaged (extract) | 0.60 |
   | Wasabi | 0.25 |
   | Horseradish (extract) | 0.52 |
   | Ascorbic acid | 112 |
   | Cacao (extract) | 167 |
   | Black pepper (extract) | 0.30 |
   | Cannabis (extract) | 25 |
2. Add 100 µL of the extract or stock and 100 µL *E. coli* culture with an OD600 of 0.1 to a 96 wells plate. A negative control of 100 µL milliQ must be included in the measurement
3. Incubate plate in a microplate incubator shaker at 37 °C at 600 rpm
4. Take OD600 measurements every 30 minutes until the OD of the negative control reaches 0.5
5. Determine relative growth compared to the negative control

MIC determination

1. Prepare stocks of serial dilutions in antibiotics in LB
2. Add 100 µL antibiotic stock solution and 100 µL *E. coli* culture with an OD of 0.1 to a 96 wells plate
3. Incubate plate in a microplate incubator shaker at 37 °C at 600 rpm
4. Take OD600 measurements every 30 min until the OD of the negative control reaches 0.5
5. Determine the minimal concentration at which growth is inhibited

[NotebookClick here to visit our Notebook to see which experiments we did when and how they turned out.](https://2018.igem.org/Team%3ALeiden/Notebook)
[PartsClick here to visit our Parts page and see which BioBricks we have constructed and characterized.](https://2018.igem.org/Team%3ALeiden/Parts)
[SafetyClick here to visit our Safety page and learn about everything concerning safety in our project.](https://2018.igem.org/Team%3ALeiden/Safety)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Leiden/Results

Although the mobile version has the same content, this wiki is best viewed on larger screens

Loading a bunch of figures...

# Results

The world is in sore need of new antibiotics. [Read more about the issue of antibiotic resistance on our Project Description page](https://2018.igem.org/Team%3ALeiden/Description) Antibiotic resistance is an ever growing problem and alarmingly few novel antibiotic
therapies have been developed in the past decades[1]. Based on  [Read more about our discussions with these experts
on our Integrated Human Practices page](https://2018.igem.org/Team%3ALeiden/Human_Practices) conversations with experts in the field of antibiotic discovery,
we determined two key missed opportunities in antibiotic discovery research. Firstly, current screening efforts suffer from tunnel
vision: only the lethality of compounds is considered. This overlooks a potential goldmine of compounds that are not lethal, but
stressful. Stressful compounds can be used in synergistic combination therapies that enhance current antibacterial treatments or
create entirely novel ones. Moreover, combination therapies could delay the onset of antibiotic resistance because multiple resistance
pathways are required to combat these therapies[2]. A second missed opportunity is the early determination of information about the
drugs mode of action, which can aid in shortening drug discovery pipelines.

With these considerations in mind, we aimed to develop a screening platform capable of detecting specific bacterial cell stresses.
For this, we created a library of reporter strains using promoters of genes involved in stress pathways. We made two types of reporter
strains: 1) GFP reporters which can be quantitatively analysed using flow cytometry or plate reader measurements, and 2) visual reporters
that use chromoproteins to create a readout visible to the naked eye. Visual reporters can be used in experiments where qualitative analysis
is cumbersome or not essential, such as overlays or disk diffusions. Furthermore, visual reporters can be used in labs where expensive equipment
necessary for GFP measurements is not available.

### References

[1]: Silver, L. L. (2011). Challenges of Antibacterial Discovery. Clinical Microbiology Reviews, 24(1), 71–109. https://doi.org/10.1128/cmr.00030-10

[2]: Xu, X., Xu, L., Yuan, G., Wang, Y., Qu, Y., & Zhou, M. (2018). Synergistic combination of two antimicrobial agents closing each other’s mutant selection windows to prevent antimicrobial resistance. Scientific Reports, 8(1).
doi:10.1038/s41598-018-25714-z

Plasmid construction – Creating 26 reporter strains for the detection of cellular stress

##

To identify genes that are upregulated in response to various stresses in *E. coli*,  [To consult this literature, please
visit the
respective part pages of each promoter. A full list of our parts can be found on our parts page](https://2018.igem.org/Team%3ALeiden/Parts) literature was consulted. Next, to isolate the promoters of these genes, a 300 bp region upstream of the
stress-related genes was amplified by PCR from *E. coli* genomic DNA.  [Visit our primers page for a list of primers used](https://2018.igem.org/Team%3ALeiden/Parts)
Forward primers contained EcoRI, XbaI overhangs and reverse primers contained PstI and SpeI overhangs, in order to create parts in the BioBrick format (Figure 1). Visit our primers page for a list of primers used. The
promoter fragments were subsequently ligated into chloramphenicol backbones ([pSB1C3](http://parts.igem.org/Part%3ApSB1C3)). See figure 2 for an overview of all promoters and their stress
pathway of origin. To ensure future teams and other researchers will be able to use our work, we submitted a total of 26 stress-related promoters and one novel constitutive promoter (see
the chromoprotein section) as
 [Visit our Parts to see all the parts we submitted to the iGEM registry](https://2018.igem.org/Team%3ALeiden/Parts) basic parts to the iGEM part registry.

![](https://static.igem.org/mediawiki/2018/e/e8/T--Leiden--Results-Fig1.jpg)

**Figure 1. Amplification of genomic stress-related promoters.** 300 bp regions of upstream of stress related genes in the *E. coli* genome were amplified by PCR and used for cloning of the reporter constructs. Not all
amplifications were successful as in the case of pAcpP.

##

![](https://static.igem.org/mediawiki/2018/d/d7/T--Leiden--Description-StressPromotors.png)

**Figure 2. Overview of promoters isolated from the *E. coli* genome and stress pathways of origin**

For the creation of reporter strains, our isolated promoters were cloned upstream of [GFP](http://parts.igem.org/Part%3ABBa_E0040),
[mCherry](http://parts.igem.org/Part%3ABBa_J06504),
[AmilCP](http://parts.igem.org/Part%3ABBa_K592009) (blue chromoprotein) or
[spisPink](http://parts.igem.org/Part%3ABBa_K1033932) (pink chromoprotein) reporter genes. Cloning was performed using the
 [Visit our Protocols page for more information](https://2018.igem.org/Team%3ALeiden/Experiments) BioBrick assembly method. Correct insertion was verified
by restriction analysis with NdeI and PvuI, see figure 3. 18 of these constructs were submitted as
 [Visit our Parts page to view all the parts we submitted to the iGEM registry](https://2018.igem.org/Team%3ALeiden/Parts) composite parts.

![](https://static.igem.org/mediawiki/2018/9/9d/T--Leiden--Results-Fig3.png)

**Figure 3. Construction of reporter plasmids using the BioBrick system.** A) Plasmid map of a
typical stress reporter plasmid. Shown here is the [pSB1C3](http://parts.igem.org/Part%3ApSB1C3) plasmid containing the promoter of recN followed by the gfp gene,
yielding a construct of 3169 bp. B). The restriction analysis with NdeI and PvuI used to confirm correct insertion of the promoters.
Shown are the constructs with the promoters of umuC, soxS, secA, relA, recN, and recA.

## Conclusion

26 promoter regions of stress-related genes and one novel constitutive promoter were successfully
amplified from the *E. coli* genome, ligated into the chloramphenicol backbone ([pSB1C3](http://parts.igem.org/Part%3ApSB1C3)) and submitted as BioBrick basic parts.
These promoters were used to create reporter strains with GFP, mCherry, AmilCP and spisPink as reporter genes, 18 of which were submitted as BioBrick
 [Visit our Parts to see all the parts we submitted to the iGEM registry](https://2018.igem.org/Team%3ALeiden/Parts)  composite parts.

Validation of stress-induced GFP expression.

## Characterising our promoter repertoire

The goal of our system is to enable detection of specific types of cellular stress using reporter strains.
To this end, we performed characterisation experiments of our GFP reporter strains using flow cytometry. To establish which reporter strains could successfully detect different
types of cellular stresses, we incubated cells with a range of stressors with different targets in the cell, see table 1 for an overview.

**Table 1. Stress pathways targeted and stressors used in the initial characterisation of our GFP reporter strains.**

| Stress pathway | Stressor |
| Oxidative stress | Hydrogen peroxide |
| Envelope stress | Ethanol |
| DNA damage | Nalidixic acid, zeocin |
| RNA synthesis | Rifampicin |
| Protein synthesis | Apramycin, chloramphenicol, hygromycin, kanamycin, streptomycin, tetracycline |
| Cell wall synthesis | Ampicillin, carbenicillin, vancomycin |

After measuring using flow cytometry, results of this preliminary screening were summarised in a heatmap showing relative expression levels which were normalised to incubation without stressors (Figure 4).
For a detailed description of the measurement process, see our [Protocols](https://2018.igem.org/Team%3ALeiden/Experiments) and
[Measurement](https://2018.igem.org/Team%3ALeiden/Measurement) pages.

![](https://static.igem.org/mediawiki/2018/c/ce/T--Leiden--Results-Heatmap.png)

**Figure 4. Heatmap showing relative GFP expression after exposure to stressors.** Values for each promoter are normalised to its respective negative control (no stressor, not shown). Appendix A shows the values with
standard deviation and number of experiment replications.
*E. coli* DH5α is included as a negative control to measure the autofluorescence of cells. For a detailed description of the measurements and
experimental setups, see the [Protocols](https://2018.igem.org/Team%3ALeiden/Experiments) and
[Measurement](https://2018.igem.org/Team%3ALeiden/Measurement) pages.

Our heatmap shows large variation in the response of promoters to our library of stressors, thereby showcasing the potential of a system utilizing
stress promoters to determine and distinguish bacterial stress types. Of the 24 promoters we tested, 8 promoters showed significant upregulation in
response to one or a few stressors from our utilized library, shown in table 2. Promoters with no significant response will need further validation
using additional stressors to fully exploit their use.

| Reporter construct | BioBrick code | Inducing stressor |
| --- | --- | --- |
| pKatG-GFP | [BBa\_K2610018](http://parts.igem.org/Part%3ABBa_K2610018) | H2O2 |
| pRelA-GFP | N/A | Carbenicillin, streptomycin |
| pSoxS-GFP | [BBa\_K2610031](http://parts.igem.org/Part%3ABBa_K2610031) | H2O2, nalidixic acid |
| pSecA-GFP | [BBa\_K2610011](http://parts.igem.org/Part%3ABBa_K2610011) (promoter only) | Kanamycin |
| pRpoE-GFP | [BBa\_K2610021](http://parts.igem.org/Part%3ABBa_K2610021) (promoter only) | Ethanol, chloramphenicol, nalidixic acid |
| pSpy-GFP | [BBa\_K2610025](http://parts.igem.org/Part%3ABBa_K2610025) (promoter only) | Ethanol, chloramphenicol, nalidixic acid |
| pInaA-GFP | [BBa\_K2610026](http://parts.igem.org/Part%3ABBa_K2610026) (promoter only) | H2O2, nalidixic acid |
| pClpB-GFP | [BBa\_K2610020](http://parts.igem.org/Part%3ABBa_K2610020) | Ethanol |

## Validating our promoters using confocal microscopy

The pSoxS-GFP ([BBa\_K2610031](http://parts.igem.org/Part%3ABBa_K2610031)) BioBrick was also validated using confocal microscopy. Incubation of our reporter strain with
nalidixic acid can be seen
to increase GFP expression compared to basal GFP expression levels (Figure 5).

![](https://static.igem.org/mediawiki/2018/e/e0/T--Leiden--Results-Fig5.png)

**Figure 5. Visualisation of GFP expression of the pSoxS-GFP ([BBa\_K2610031](http://parts.igem.org/Part%3ABBa_K2610031)) strain during nalidixic acid treatment
using confocal microscopy.**
Wild-type DH5α and the pSoxS-GFP ([BBa\_K2610031](http://parts.igem.org/Part%3ABBa_K2610031)) strain were incubated with nalidixic acid (50 ng/mL) for two hours.
A/B)
Wild-type DH5α.
C/D) pSoxS-GFP without stressor. Basal GFP expression can be observed. E/F) pSoxS-GFP with 50 ng/mL nalidixic acid. An increase in expression
compared to the negative control can be observed. Pictures were obtained by using a confocal microscope. The scale bar represents 10 µm.

One of our BioBricks, pCspA-GFP ([BBa\_K2610037](http://parts.igem.org/Part%3ABBa_K2610037)), showed a significant downregulation as a specific response to all protein
synthesis inhibitors in our
library. It should be noted that this response could be caused by inhibition of GFP production due to the protein synthesis stressors.
Similarly, in our [Measurement](https://2018.igem.org/Team%3ALeiden/Measurement) page we show that protein synthesis inhibitors lead to lower observed GFP expression for constitutive promoters,
the same could be occurring with pCspA which has a high basal expression level.

However, we still believe this result to be relevant as other promoters show no apparent specific downregulation by protein synthesis inhibitors only. Furthermore, we validated this construct further using confocal microscopy (Figure 6).
We induced the pCspA-GFP ([BBa\_K2610037](http://parts.igem.org/Part%3ABBa_K2610037))
BioBrick with kanamycin and included a pSoxS-mCherry ([BBa\_K2610032](http://parts.igem.org/Part%3ABBa_K2610032)) as a positive control for protein synthesis. Although the
base-level signal of mCherry is much lower than that of GFP, we can see that GFP expression is selectively inhibited when the strains are treated with kanamycin while mCherry is not.
Whether the culprit of decreased GFP expression is protein synthesis inhibition or promoter regulation could be confirmed further in the future by using a *Tet-On Tet-Off* system[3]. Here, downregulation of pCspA would
cause an increase in GFP expression. A response of pCspA due to protein synthesis inhibitors could then be confirmed by the observation that incubation with such agents would lead to an increase in GFP production.

![](https://static.igem.org/mediawiki/2018/8/83/T--Leiden--Results-Fig6.png)

**Figure 6. Selective downregulation of GFP expression in the pCspA-GFP strain.**
A coculture of pCspA-GFP ([BBa\_K2610037](http://parts.igem.org/Part%3ABBa_K2610037)) and pSoxS-mCherry ([BBa\_K2610032](http://parts.igem.org/Part%3ABBa_K2610032))
was incubated for two hours with ampicillin (50 µg/mL), nalidixic acid (50 ng/mL) and a combination of nalidixic acid and kanamycin (50 µg/mL). A) Treatment with ampicillin as a
negative control. A high base level of GFP expression from the pCspA-GFP strain can be observed. B) Treatment with nalidixic acid for induction of pSoxS-mCherry showed no significant increase in mCherry expression. C) Treatment with
nalidixic acid and kanamycin decreases expression of GFP while mCherry levels are unaffected.

## Determining promoter dose dependency

We validated the GFP reporters pKatG ([BBa\_K2610018](http://parts.igem.org/Part%3ABBa_K2610018)), pSoxS ([BBa\_K2610031](http://parts.igem.org/Part%3ABBa_K2610031)),
pSpy ([BBa\_K2610025](http://parts.igem.org/Part%3ABBa_K2610025)), pRpoE ([BBa\_K2610021](http://parts.igem.org/Part%3ABBa_K2610021))
and pInaA ([BBa\_K2610026](http://parts.igem.org/Part%3ABBa_K2610026)) further with a dose-dependence study using the stressors they were originally found to respond
strongest to, the results of which are shown in figure 7. For all promoters, we see a clear dose-dependent response. Our pSoxS-GFP ([BBa\_K2610031](http://parts.igem.org/Part%3ABBa_K2610031))
BioBrick in particular displays the power of our system. An increase of GFP expression can be observed at a concentration 20 times lower than the observed lethal dose.

![](https://static.igem.org/mediawiki/2018/f/f9/T--Leiden--Results-Fig7D.png)

![](https://static.igem.org/mediawiki/2018/5/52/T--Leiden--Results-Fig7E.png)

![](https://static.igem.org/mediawiki/2018/0/02/T--Leiden--Results-Fig7B.png)

![](https://static.igem.org/mediawiki/2018/7/7b/T--Leiden--Results-Fig7A.png)

![](https://static.igem.org/mediawiki/2018/9/9d/T--Leiden--Results-Fig7C.png)

**Figure 7. Dose-dependence study of selected reporter constructs.** GFP reporters of pKatG, pSoxS, pSpy, pRpoE and pInaA were treated with increasing concentrations of the stressors shown to induce these promoters in the
preliminary screening (figure 4). A decrease in detected GFP expression at higher concentrations can be attributed to lethality of the stressor.

## Conclusion

Flow cytometry measurements with promoter-GFP constructs show that reporter strains for different types of cellular stress can be selectively and dose-dependently induced by different types of stressors. The selectivity in particular
establishes an important proof of concept for our system, as this shows that multiple reporters could theoretically be combined to detect different stresses in a single cell line. Of 26 promoters, 8 showed a significant response to our
library of stressors. More research is needed to discover inducing compounds for the remaining reporter strains.

### References

[3]: Baron, U., & Bujard, H. (2000). Tet repressor-based system for regulated gene expression in eukaryotic cells: Principles and advances. Methods in Enzymology, 327, 401–421. https://doi.org/10.1016/s0076-6879(00)27292-3

Signal amplification

##

We have established that our stress reporters can be used to detect cell stress at stressor concentrations much lower than the lethal dose.
To improve these results, we attempted to increase the dose response GFP expression even further, to allow for detection of even lower
concentrations of stressful substances. Additionally, increased signal intensity would facilitate GFP-based stress detection with less sensitive
detection methods. Furthermore, a readout may be obtained in a shorter time period, which would increase throughput of compound screening.
This amplification method could also be applied to our visual reporters based on chromoproteins (discussed further in the visual stress
detection system section).
Although we have shown our chromoprotein stress reporters work, the readout is not easily visible and only occurred after two to three days.

We attempted to amplify the reporter signal in two ways. First of all, we tried to create a T7 RNA amplification system. For this, we coupled stress promoters to the T7 RNA Polymerase gene ([BBa\_K2610007](http://parts.igem.org/Part%3ABBa_K2610007)).
This should result in the T7 RNA Polymerase being produced in response to stress. Next, we coupled a T7 Promoter ([BBa\_K2610005](http://parts.igem.org/Part%3ABBa_K2610005))
- which can only be read by the T7 RNA Polymerase -
to visual reporters. Unfortunately, we were not able to validate a working T7 enhancement system. Despite this, we have submitted our pSoxS-T7 RNA Polymerase ([BBa\_K2610028](http://parts.igem.org/Part%3ABBa_K2610028))
and pT7-GFP ([BBa\_K2610029](http://parts.igem.org/Part%3ABBa_K2610029)) BioBricks to the registry in order to give others the
opportunity to test this system.

Thereafter, we tried increasing the amount of promoter regions and GFP genes on one plasmid, see table 3 for the constructs used.

**Table 3. Constructs used for signal amplification**

| Construct | BioBrick code |
| pSoxS-GFP | [BBa\_K2610031](http://parts.igem.org/Part%3ABBa_K2610031) |
| pSoxS-GFP-pSoxS-GFP | [BBa\_K2610034](http://parts.igem.org/Part%3ABBa_K2610034) |
| pSoxS-GFP-GFP-pSoxS-GFP-GFP | [BBa\_K2610035](http://parts.igem.org/Part%3ABBa_K2610035) |

Using these amplified BioBricks, a nalidixic acid dose-dependency study was performed.
A clear increase in both absolute and relative signals compared to the negative controls can be observed in the amplified BioBricks (pSoxS-GFP-pSoxS-GFP and pSoxS-GFP-GFPpSoxS-GFP-GFP), compared to pSoxS-GFP (Figure 8). This shows that
the GFP signal can be amplified by simple multiplication of promoter regions and GFP genes on one plasmid.

![](https://static.igem.org/mediawiki/2018/f/fa/T--Leiden--Results-Fig8A.png)

![](https://static.igem.org/mediawiki/2018/3/3c/T--Leiden--Results-Fig8B.png)

![](https://static.igem.org/mediawiki/2018/0/09/T--Leiden--Results-Fig8C.png)

**Figure 8. Amplification of the signal obtained from the pSoxS-GFP strain.** Increasing promoter regions and GFP genes of our pSoxS-GFP reporter is shown to increase both absolute and relative signals after treatment with
nalidixic acid. These measurements were performed in the same flow cytometry session so that mean fluorescence intensity (MFI) values can be confirmed.

A disadvantage of this multiplication is that it also increases the basal expression level. Nevertheless, we observe an increase in relative expression for the amplified BioBricks, meaning they are still beneficial. In the future, we hope
to increase signal strength further and keep the GFP expression as low as possible when the strain is not stressed. Further research into the T7-RNA-polymerase amplification system could provide the answer to this challenge.

## Conclusion

We successfully amplified the pSoxS-GFP stress reporter strain by increasing the amount of promoters and GFP genes in the plasmid. This enables detection of even lower concentrations of stressful substances, allows signals to be detected
by
less sensitive detection devices and facilitates faster signal detection. When a visual reporter such as a chromoprotein is used, the time to obtain a significant readout may also be reduced.

Validating our system for non-antibiotic compounds

##

With earlier experiments, we have shown that our system can be used to detect lethal substances in low - stressful - concentrations. In our final product, we want to be able to detect non-lethal stressful substances for use in synergistic
combination therapies with other stressful substances. In this chapter, we show that our system can be used to detect stress caused by non-lethal compounds which are capable of enhancing the effect of antibiotics.

## Verification of non-lethality

To be able to test non-lethal stressful substances, we first had to ensure substances we were using were not lethal. To that end, we tested 19 natural compounds, or extracts thereof, available in the supermarket and sometimes heralded in
popular culture for their medicinal properties. We first assessed the growth of *E. coli* following addition of the potential stressors compared to the addition of water. Three of these compounds were found to be lethal in the
concentration
used, as seen in figure 9. Due to time constraints, we decided to test three compounds which did not shows lethality and growth inhibitions, and we thought were interesting based on literature. These three substances are ascorbic acid[4][5],
cannabis[6][7] and ginger[8][9].

![](https://static.igem.org/mediawiki/2018/6/66/T--Leiden--Results-Fig9.png)

**Figure 9. Assessment of lethality of 19 products available in the supermarket.** Wild-type DH5α was incubated with natural products and OD was measured in a plate reader. The growth was normalised to the negative control
(addition of milliQ).

## Stress induction of non-lethal compounds

Extracts of ginger and cannabis and a vitamin C solution were incubated with GFP reporter strains containing promoters of the following genes: *dnaK, cutF, clpB, rseA, cspA, relA, and nusA*, results of which are shown in figure 10.
These promoters were chosen for two reasons: 1) they originate from a broad spectrum of stress pathways and 2) we wanted to validate promoters that did not show a significant response in the preliminary screening (with the exception of
clpB).

![](https://static.igem.org/mediawiki/2018/9/98/T--Leiden--VITCclpBresult.png)

**Figure 10. pClpB is induced by ascorbic acid.**  7 promoter-GFP strains were incubated with ascorbic acid, ginger and cannabis. Only pClpB-GFP ([BBa\_K2610020](http://parts.igem.org/Part%3ABBa_K2610020))
showed a significant response to ascorbic acid.

The highest response was seen for ascorbic acid with the clpB promoter. We therefore decided to continue with ascorbic acid and performed a dose-dependence study. To exclude the possibility of stress occurring due to the acidity of
ascorbic acid, we included pH controls. These were determined by measuring the pH of ascorbic acid solutions in LB and bringing fresh LB samples to the same pH using concentrated HCl. The results are shown in figure 11.

![](https://static.igem.org/mediawiki/2018/d/d8/T--Leiden--Results-Fig11.png)

**Figure 11. Dose-dependency of pClpB-GFP ([BBa\_K2610020](http://parts.igem.org/Part%3ABBa_K2610020)) induction by ascorbic acid.** Ascorbic acid
concentrations are denoted in mg/mL. A significant stress response higher than the pH control was observed for incubation with 2.5 and 10 mg/mL ascorbic acid.

Ascorbic acid is seen to induce significant stress not observed in the pH control for the clpB promoter at a concentration of 2.5 mg/mL. However, the GFP response decreases sharply at 5 mg/mL and rises again at 10 mg/mL. This is contrary
to expectations, as we expected to see a dose response, similar to figure 7. Unfortunately, due to time constraints we were unable to repeat the experiment to rule out an experimental error. Still, based on the results in figures 9 and 10
we can draw the cautious conclusion that ascorbic acid induces the clpB promoter and is stressful at these concentrations.

Having confirmed that ascorbic acid induces cellular stress, we set out to confirm its synergistic use in combination with antibiotics. The MIC values of the antibiotics were determined with and without the addition of ascorbic acid
(Figure 12). A clear drop in the MIC value was observed for ampicillin, chloramphenicol and nalidixic acid, up to 82% in the case of chloramphenicol. This is a
remarkable result considering these three antibiotics all have different modes of action and ascorbic acid is apparently able to enhance all of them.

![](https://static.igem.org/mediawiki/2018/4/43/T--Leiden--DemonstrateFig6A.png)

![](https://static.igem.org/mediawiki/2018/5/56/T--Leiden--DemonstrateFig6B.png)

![](https://static.igem.org/mediawiki/2018/8/8c/T--Leiden--DemonstrateFig6C.png)

![](https://static.igem.org/mediawiki/2018/7/7c/T--Leiden--Results-Fig12D.png)

![](https://static.igem.org/mediawiki/2018/2/25/T--Leiden--Results-Fig12E.png)

![](https://static.igem.org/mediawiki/2018/4/4b/T--Leiden--Results-Fig12F.png)

**Figure 12. Addition of ascorbic acid dramatically reduces MIC values of selected antibiotics.**  The MIC values of ampicillin, apramycin, chloramphenicol, nalidixic acid, tetracycline and streptomycin were determined with
and without the addition of ascorbic acid (5 mg/mL).

## Conclusion

In the future, we intend our stress detection system to be used for discovery of non-lethal compounds that induce cellular stress. These substances can synergistically enhance current antibiotics or be used in novel combination therapies.
We have shown that ascorbic acid is an ideal candidate compound. First, we verified its non-lethality. Next, we observed that it induced the *clpB* promoter showing that it is a stressful compound. Finally, we proved that ascorbic
acid enhances the effect of ampicillin, chloramphenicol and nalidixic acid. These experiments demonstrate that our system is capable of detecting stressful compounds, which can be used in synergistic combination therapies.

### References

[4] Tajkarimi, M., & Ibrahim, S. A. (2011). Antimicrobial activity of ascorbic acid alone or in combination with lactic acid on Escherichia coli O157:H7 in laboratory medium and carrot juice. Food Control, 22(6), 801–804.
https://doi.org/10.1016/j.foodcont.2010.11.030

[5] Mathew, S., Verghese, R., & David, A. (2017). Antimicrobial activity of Vitamin C demonstrated on uropathogenic Escherichia coli and Klebsiella pneumoniae. Journal of Current Research in Scientific Medicine, 3(2), 88.
https://doi.org/10.4103/jcrsm.jcrsm\_35\_17

[6] Ali, E. M. M., Almagboul, A. Z. I., Khogali, S. M. E., & Gergeir, U. M. A. (2012). Antimicrobial Activity of Cannabis sativa L. Chinese Medicine, 03(01), 61–64. https://doi.org/10.4236/cm.2012.31010

[7] Chakraborty, S., Afaq, N., Singh, N., & Majumdar, S. (2018). Antimicrobial activity of Cannabis sativa, Thuja orientalis and Psidium guajava leaf extracts against methicillin-resistant Staphylococcus aureus. Journal of
Integrative Medicine, 16(5), 350–357. https://doi.org/10.1016/j.joim.2018.07.005

[8] Patel, R. V., Thaker, V. T., & Patel, V. (2011). Antimicrobial activity of ginger and honey on isolates of extracted carious teeth during orthodontic treatment. Asian Pacific Journal of Tropical Biomedicine, 1(1),
867–871. https://doi.org/10.1016/s2221-1691(11)60124-x

[9] Gupta, S., & Ravishankar, S. (2005). A Comparison of the Antimicrobial Activity of Garlic, Ginger, Carrot, and Turmeric Pastes Against Escherichia coli O157:H7 in Laboratory Buffer and Ground Beef. Foodborne Pathogens
and Disease, 2(4), 330–340. https://doi.org/10.1089/fpd.2005.2.330

A visual stress detection system

## A new positive control - Isolation and characterisation of a novel constitutive promoter

During our project, we isolated the GapA promoter ([BBa\_K2610040](http://parts.igem.org/Part%3ABBa_K2610040)) from the *E. coli* genome. Contrary to our stress
promoters,
this promoter has a constitutive high basal expression level, as can be seen in the spun down cultures of our pink and blue chromoprotein BioBricks:
pGapA-AmilCP ([BBa\_K2610041](http://parts.igem.org/Part%3ABBa_K2610041)) and pGapA-spisPink ([BBa\_K2610042](http://parts.igem.org/Part%3ABBa_K2610042)),
Figure 13. In contrast to many synthetic promoters, this novel promoter provides an indication of high levels of *natural* expression. Therefore, this promoter can be used as a future positive control of reporter production - such as
GFP - in a natural situation.

![](https://static.igem.org/mediawiki/2018/4/4c/T--Leiden--Results-Fig13.png)

**Figure 13. Pelleted liquid cultures of pGapA-chromoprotein strains show high levels of expression.**  The shown pellets were spun down from 50 mL of overnight culture.

## Chromoprotein-based stress detection for overlays and disk diffusion

Experiments in which potential antibiotic producing strains or disks containing antibiotics are placed on a plate, after which a bacterial test strain is
seeded on top.
Compounds
diffuse through the agar and - if lethal - prevent bacterial growth surrounding the strain or disk, thereby creating a visible “zone of inhibition" Overlays and disk diffusions have been pivotal in the past for
the discovery and characterisation of antibiotics[10][11]. Because these experiments are carried out on agar plates, a GFP-based readout is not suitable, since GFP production would be cumbersome to measure reproducibly on a
plate. Furthermore, quantitative measurements are not essential in this case, as these techniques are purely used to identify interesting compounds or antibiotic producing strains. Therefore, we chose for a
 [Read more about this decision on our Integrated Human Practices page](https://2018.igem.org/Team%3ALeiden/Human_Practices) readout visible to the naked
eye in the form of chromoproteins.
Disk diffusion and overlay experiments display halos where bacteria cannot grow. At the edge of such a halo, there is a zone with a [Read more about this zone, and the determination of it on our Model page](https://2018.igem.org/Team%3ALeiden/Model)  non-lethal antibiotic concentration. In this zone we could potentially detect cellular stress. To test this, we created a plasmid
containing a blue chromoprotein regulated by a stress promoter, pSoxS-AmilCP ([BBa\_K2610033](http://parts.igem.org/Part%3ABBa_K2610033)) and pGapA-spisPink ([BBa\_K2610033](http://parts.igem.org/Part%3ABBa_K2610033)). From our flow cytometry experiments, we knew that pSoxS should react to nalidixic acid. We attempted a disk diffusion with our pSoxS-AmilCP BioBricks, which is shown in figure 14. A
discoloration around the halo can be seen, which is not seen in the negative control. This shows we can visualize bacterial cell stress using chromoproteins.

![](https://static.igem.org/mediawiki/2018/7/76/T--Leiden--Results-Fig14A.png)

![](https://static.igem.org/mediawiki/2018/b/bd/T--Leiden--Results-Fig14B.png)

**Figure 14. Cellular stress can be visualised on agar-plates using chromoprotein reporter strains.**  Disk diffusions with nalidixic acid were carried out using wild-type DH5α and our pSoxS-AmilCP ([BBa\_K2610033](http://parts.igem.org/Part%3ABBa_K2610033)) strain.
A) A faint discolouration around the halos in the right agar plate can be observed which is indicative of non-lethal cellular stress. B) Disk diffusion with pSoxS-AmilCP ([BBa\_K2610033](http://parts.igem.org/Part%3ABBa_K2610033)) and nalidixic acid. Two eppendorf tubes containing the pSoxS-AmilCP ([BBa\_K2610033](http://parts.igem.org/Part%3ABBa_K2610033))
strain (left) and a wild-type DH5α (right) are shown for reference. Again, a darker colour around
the halo is seen in the disk diffusion.

We also attempted to achieve similar results in liquid cultures by incubating bacteria expressing the pSoxS-AmilCP BioBrick with increasing concentrations of nalidixic acid, results of which are shown in figure 15. However, we found no
discernible difference in color for the stressed vs. unstressed strains. Evidently, the stress caused by nalidixic acid at our used concentrations is not enough to create a difference in expression visible to the naked eye. Further
optimisation with, among other things, different incubation times is necessary to achieve the desired results.

![](https://static.igem.org/mediawiki/2018/3/3b/T--Leiden--Results-Fig15.jpg)

**Figure 15. Attempted dose-response in chromoprotein liquid cultures.** pSoxS-AmilCP ([BBa\_K2610033](http://parts.igem.org/Part%3ABBa_K2610033)) strains
were stressed overnight with four concentrations of nalidixic acid. No dose-dependent response was observed.

## Conclusion

We isolated a novel constitutive promoter from the *E. coli* genome and showed a high natural expression level of chromoproteins. Additionally, we have shown that chromoproteins can be used as visual reporters for cell stress in
conditions
representative of overlay and disk diffusion experiments. This is the first time visual reporters have been used in such a way. This opens the door for many agar plate-based detection systems. In addition, visual reporters can be used in
labs where expensive plate readers and flow cytometers that measure GFP expression are not available. Since our BioBricks are available on the registry, experiments using these strains can begin today!

### References

[10]: Balouiri, M., Sadiki, M., & Ibnsouda, S. K. (2016). Methods for in vitro evaluating antimicrobial activity: A review. Journal of Pharmaceutical Analysis, 6(2), 71–79. https://doi.org/10.1016/j.jpha.2015.11.005

[11]: Lewis, K. (2013). Platforms for antibiotic discovery. Nature Reviews Drug Discovery, 12(5), 371–387. https://doi.org/10.1038/nrd3975

Conclusion and future perspectives

## Conclusion

The global challenge of antibiotic resistance requires novel approaches in the field of antibiotic discovery. Two major missed opportunities in antibiotic research are the detection of non-lethal stressful compounds and the early
determination of a compound’s mode of action. With Fifty Shades of Stress, we tackle both issues.

To achieve this, we isolated promoters of stress-related genes from the *E. coli* genome using PCR and submitted 27 of them as [Read
about each of our parts on our Parts page](https://2018.igem.org/Team%3ALeiden/Parts) basic parts to the registry.
The promoters were cloned upstream of two fluorescent reporter genes, [GFP](http://parts.igem.org/Part%3ABBa_E0040) and [mCherry](http://parts.igem.org/Part%3ABBa_J06504),
and two visual reporter genes (chromoproteins), [amilCP](http://parts.igem.org/Part%3ABBa_K592009) (blue) and [spisPink](http://parts.igem.org/Part%3ABBa_K1033932)
(pink). 18 of these constructs were submitted as
 [Read about each of our parts on our Parts page](https://2018.igem.org/Team%3ALeiden/Parts) composite parts.

To characterize the stress responsiveness of our promoters, we subjected 24 GFP reporter strains to a library of stressors that elicit different
types of stress. 8 reporters yielded a clear increase in GFP expression in response to one or more compounds in our library. Several were
validated further using confocal microscopy and/or dose-response studies. Additionally, one reporter, pCspA-GFP ([BBa\_K2610037](http://parts.igem.org/Part%3ABBa_K2610037)),
was presumably selectively downregulated, although more research is needed to confirm this.

To improve signal detection, we have amplified GFP expression by increasing the number of promoters and GFP genes in one plasmid. We performed the experiment using the pSoxS ([BBa\_K2610030](http://parts.igem.org/Part%3ABBa_K2610030)) BioBrick and the signal increases almost tenfold. These parts were also submitted as composite parts.

To prove that our system could be used to detect stressful compounds, we tested a range of natural products for their lethality. Three of these compounds were tested on different stress promoters and ascorbic acid was found to be
stress-inducing. Therefore, ascorbic acid was incubated together with several antibiotics and the MIC value was found to drop drastically, up to 82% for ampicillin. Thereby proving our system is capable of detecting stressful compounds
suitable for antibiotic combination therapies.

For experiments with antibiotics on agar plates - such as disk diffusions or overlays - we set out to create a visual reporter strain using chromoproteins, an approach which has not been attempted before. We performed a disk diffusion
experiment using our pSoxS-AmilCP ([BBa\_K2610033](http://parts.igem.org/Part%3ABBa_K2610033)) BioBrick as a reporter and found that a discoloration occurred in the area
where the antibiotic concentration is non-lethal. This is an important proof of concept for visual reporters to be used in agar-based experiments.

## Towards an application

We have shown that the foundation for Fifty Shades of Stress is solid. The next step is creating an easy-to-use application from our project. To that end, we have created a  [Read about our product design in detail on our Product Design wiki page](https://2018.igem.org/Team%3ALeiden/Applied_Design) product design for an easy-to-use bacterial cell stress detection kit with the name 50S.O.S.

We want to establish a proof of concept for three important aspects of the kit before full scale production can begin. First, we want GFP expression by our reporter strains to be measurable in plate readers. This would make measurement
faster and open up the possibility of obtaining real-time data. Unfortunately, we experienced repeated complications when measuring in the plate reader available to us, therefore preventing us from examining this possibility further.
However, quantifying GFP expression in *E. coli* strains using a plate reader is not new and should work[12]. We surmise that the cause of our failed plate reader experiments may be unsuitable transparent plates or diluting
the cultures too much before stressing them.

A second goal for our future application is to enable testing of multiple stress promoters in a single bacterial strain, by using fluorescent reporters with different emission wavelengths. We attempted this with the GFP and mCherry
reporters using co-transformations and by combining two reporter constructs in one plasmid. However, neither technique yielded a replication of the results obtained with the single-promoter GFP or mCherry plasmids. This may be due to a
lower basal expression level of mCherry or lower fluorescence intensity. Still, we submitted two combined plasmids as composite parts for other teams and researchers to use and improve: pSoxS-mCherry-pKatG-GFP ([BBa\_K2610043](http://parts.igem.org/Part%3ABBa_K2610043)) and pSoxS-mCherry-pMicF-GFP ([BBa\_K2610044](http://parts.igem.org/Part%3ABBa_K2610044)). Also, we do not foresee
any fundamental reason why our combination plasmids should not have worked[13], so we are confident further research could easily allow for the combinations of multiple stress reporters in a single bacterial strain.

A third important aspect of our 50S.O.S. kit is the use of freeze dried strains, to allow for safer GMO transport. This is a common technique and should work according to literature. We have performed such freeze drying experiments with
our strains using  [More information can be found on our Protocols page](https://2018.igem.org/Team%3ALeiden/Experiments) glycerol.
Although we were able to revive our strains when using high glycerol concentrations, reviving was no longer successful when using lower concentrations of glycerol. Therefore, we believe future attempts should use sucrose, which is more
commonly used for freeze drying *E. coli*[14].

Although we did not yet achieve success with these three extra aspects for our kit design, the techniques described above all have precedents in literature. That means it is simply a matter of time before they can all be integrated into a
full-fledged 50S.O.S. system. For now, researchers can use each of our publicly available stress detection strains by obtaining them from the iGEM registry. This way, we are one step closer to discovering novel antibiotic therapies and
solving the antibiotic resistance crisis.

### References

[12] Srivastava, S. K., Lambadi, P. R., Ghosh, T., Pathania, R., & Navani, N. K. (2014). Genetic regulation of spy gene expression in Escherichia coli in the presence of protein unfolding agent ethanol. Gene, 548(1), 142–148.
https://doi.org/10.1016/j.gene.2014.07.003

[13] Bjørkøy, G., Lamark, T., Pankiv, S., Øvervatn, A., Brech, A., & Johansen, T. (2009). Chapter 12 Monitoring Autophagic Degradation of p62/SQSTM1. Methods in Enzymology, , 181–197.
https://doi.org/10.1016/s0076-6879(08)03612-4

[14] Leslie, S.B., Israeli, E., Lighthart, B., Crowe, J.H., & Crowe, L.M. (1995). Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Appl Environ Microbiol, 3592-3597.

Appendix A - Heatmap data

Below the data for the heatmap with the standard error and the amount of replicates included is shown.

[MeasurementClick here to read how we ensured good measurement practices during out project](https://2018.igem.org/Team%3ALeiden/Measurement)
[DemonstrateClick here to read how we demonstrated our project](https://2018.igem.org/Team%3ALeiden/Demonstrate)
[PartsClick here to see all the parts we submitted as BioBricks](https://2018.igem.org/Team%3ALeiden/Parts)
[ModelClick here to read how we used modeling to improve our experiments](https://2018.igem.org/Team%3ALeiden/Model)