iGEM 2024 — Team anu

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Project

* [Contribution](https://2024.igem.wiki/anu/contribution)
* [Description](https://2024.igem.wiki/anu/description)
* [Engineering](https://2024.igem.wiki/anu/engineering)
* [Experiments](https://2024.igem.wiki/anu/experiments)
* [Notebook](https://2024.igem.wiki/anu/notebook)
* [Results](https://2024.igem.wiki/anu/results)

[Safety](https://2024.igem.wiki/anu/safety)
[Human Practices](https://2024.igem.wiki/anu/human-practices)

Team

* [Members](https://2024.igem.wiki/anu/team)
* [Attributions](https://2024.igem.wiki/anu/attributions)

Awards

* [Sustainable](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sustainable%27%29%20%7D%7D)
* [Education](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27education%27%29%20%7D%7D)

**Notebook**

![](https://static.igem.wiki/teams/5423/book.png)

* [dCas9](#background)
* [Golden Gate Assembly and Initial Transformations](#solution)
* [Level 2 Assembly and Transformations](#level2)
* [Combining Constructs and Verification Through Digestion](#combine)
* [Hydrogen Peroxide Experiments](#hydrogen)
* [References](#references)

## dCas9

**12/08/24- gRNA design**

Once we had decided on the dCas9 system, we used benching to design gRNA inserts to target eKatg, mScar and a non-targeting control. Plasmids were designed to minimise off target binding, whilst maintaining the protospacer adjacent motif. We added BsaI sites to the gRNA inserts, to make them compatible with the BsaI plasmid insertion.

The plasmid, and gRNA sequences were then ordered from IDT. The gRNA primers were ordered as single strands to save costs.

**19/08/24- Oligo annealing and BsmBI reaction**

When the gRNA strands arrived, they were hydrated and oligo annealed by rapidly heating, and slowly cooling, the strands in the presence of an annealing buffer.

Following annealing, the gRNAs were inserted into the pdCas9 plasmids using a BsaI golden gate reaction, which ran over night.

**20/08/24- Bacterial transformation**

Once the BsaI reaction was complete, the plasmids were transformed into host (ecoli strain) via electrocution. The cells were allowed to divide before being plated on chloramphenicol infused agar.

**21/08/24- Colony selection**

The agar plates were observed for growth, and colonies were taken and grown in LB broth with chloramphenicol. The pdCas9 plasmid contained a mRFP dropout, and as such, successful BsaI reactions were identified from the absence of red.

The plasmids contained chloramphenicol resistance genes, allowing unsuccessful transformations to be removed.

The selected colonies were then incubated overnight at 37°C.

**22/08/24 Plasmid purification, restriction enzyme digest**

gRNA plasmids were isolated from starter culture following the Isolate II Meridian low copy number protocol. Isolated plasmids were then digested with BamHI-HF and SacII, using the following conditions:

37°C for 4 hours, followed by 80°C for 20 minutes.

**23/08/24 gel electrophoresis bacterial transformation**

The digested plasmid solution was then separated by gel electrophoresis to confirm that the gRNA inserted into the plasmid backbone appropriately.

Having confirmed the successful insertion of gRNA into the plasmid backbone, we transformed E. coli K-12 with the plasmids and grew them on chloramphenicol infused agar.

**24/08/24 Attempted colony selection**

We attempted to select colonies, however the transformation was not successful

**25/08/24 bacterial transformation**

E. coli K-12 was transformed with appropriate plasmids containing gRNA.

**26/08/24 Colony selection**

The agar plates were once again observed for growth. The plates contained ecoli with the purified pdcas9 plasmid, and either a rhamnose inducible mScar plasmid, or a constitutive mScar plasmid. The presence of the pdcas9 plasmids was screened using chloramphenicol resistance. Whilst the mScar plasmids were screened via colour, and carbenicillin, and spectinomycin, respectively.

**27/08/24- Confirming cas9 function by mScarlet3 knockdown**

To confirm that the dCas9 knockdown system worked as reported by Bradley (2021), we repeated their experiment, following similar concentrations of anhydrotetracycline and arabinose. Our supervisor, Andrew George, noted that the reported optimal concentration of arabinose used by Bradley (2021) was significantly lower than what was traditionally used by others. And thus, we introduced an additional trial for a higher arabinose concentration.

**28/08/24- Fluorescence reading**

The fluorescence of the cell suspensions were corrected for optical density and compared. Levels of activity reduction did not match our guide paper. Our supervisor explained that they usually use higher arabinose concentrations then that which was listed in the paper, so we intend to attempt to survey if different levels work better.

This first test result was disheartening; however, we believe that we can still make it work of we optimization the promoters. This does become difficult, as the pdCas9 plasmid has two promoters, anhydrotetracycline for the dCas9 protein, and L-arabinose for the gRNA and its associated scaffold.

**5/09/24 – optimization**

The fluorescence reading showed that the higher L-arabinose concentration resulted in a slightly higher knockdown of mScar. We decided to repeat the mScar knockdown experiment, however we attempted to use different concentrations of L-arabinose.

**6/09/24- Fluorescence reading**

Once again, we noticed that the CRISPRi system did not result in the 40-fold reduction seen by Bradley (2021). However, the higher arabinose concentration did result in a greater knockdown of mScar.

**11/09/24 – Optimization again + starter mScar3 cultures**

Altered L-arabinose and anhydrotetracycline (aTc) concentrations to optimize CRISPRi function. Interestingly, previous results didn’t replicate showing that lower L-arabinose performed better than higher concentrations. aTc concentration had no effect on CRISPRi repression.

New pdCas9 starter cultures were developed from the MiniPrep stock.

**12/09/24 – katg crispr cell growth**

Used yesterday's starter cultures to generate 4 reaction systems:

* pdCas9 E. coli KatG + Ara + Atc
* pdCas9 E. coli KatG + Ara
* pdCas9 mScar3 + Ara + Atc
* pdCas9 mScar3 + Ara

The solutions were incubated for 2 hours, to allow them to reach 0.80 OD, at which point, H2O2 was added to the solutions. The OD was then monitored to observe for changes in the growth patterns. No changes were observed.

**13/09/24 – Sanger sequencing of primers to confirm gRNA**

The previous results have all been lackluster, so we set out to sequence our gRNA segments, to ensure that they are effectively targeting their respective proteins. We designed our PCR promoters, and ran our PCR with dideoxy nucleotides to enable us to sanger sequence the gRNAs.

Following PCR, we submitted our samples to the ANU sanger sequencing laboratory.

The results indicated that the gRNAs were perfect. This was the expected result; however, we were hopeful that the reason for our lackluster results was incorrect targeting.

We brainstormed potentially ordering new gRNAs which target the promoters of our target proteins, however this would require designing a new CRISPR system, as the pdCas9 system would not be able to target our particular protein promoters.

We did not have time to order new plasmids, and run our desired experiments, so we cut our losses and discontinued our CRISPR portion of the project.

## Golden Gate Assembly and Initial Transformations

**27/08**

Performed Golden Gate reactions for constructs 1-5 and 9-11, and transformed them into E. coli DL21 cells. The transformed cells were then plated on agar containing ampicillin to select for those that successfully incorporated the plasmids.

**28/08**

Cells expressing plasmids 9-11 failed to grow, which may have been due to a missed component in the master mix created for these reactions. We replated these failed transformations of constructs 9-11 using our existing Golden Gate mixtures.

**29/08**

Picked colonies for constructs 1-5 and 9-11 from our plates. Each selected colony was inoculated into LB broth with ampicillin to establish cultures for further analysis.

**30/08**

Conducted miniprep of constructs 1-5 and 9-11.

![](https://static.igem.wiki/teams/5423/cloning-guide-combined.png)

## Level 2 Assembly and Transformations

**2/09**

Set up level 2 Golden Gate reactions for constructs 6-8. These constructs are designed to combine elements from constructs 1-5 to create complex biosensor systems, with RFP under the control of our 3 ROS inducible promoters.

**4/09**

Transformed reactions for constructs 6-8 into E. coli DL21 cells using electroporation. The transformed cells were then plated on agar containing spectinomycin to select for those that successfully incorporated the plasmids.

**5/09**

Picked cultures for constructs 6-8 and incubated them overnight to allow for adequate growth and plasmid replication.

**6/09**

Miniprepped cultures for constructs 6-8 using a series of centrifugation and buffer steps.

## Combining Constructs and Verification through Digestion

**7/09**

Performed transformations of constructs 6-8 with constructs 9-11 to generate more integrated systems. Plated these cells on plates containing both spectinomycin and ampicillin, to select for both the catalase and biosensor plasmids. In these cells, catalase expression is inducible by rhamnose, while the ROS-inducible promoters regulate RFP.

**19/09**

Conducted overnight digests using NdeI restriction enzymes. This process involved mixing plasmid DNA with buffer, water, and the NdeI enzyme, then incubating overnight.

## Hydrogen Peroxide Experiments

**24/09**

We found that colonies containing plasmid 7 (specifically constructs 7+9, 7+10 and 7+11) exhibited extensive red fluorescence. This was unexpected given the absence of added ROS, as plasmid 7 contains the ROS-sensitive promoter Trxcp which should not lead to significant expression under these conditions. This suggests that the promoter may be leaky. Consequently, we decided to exclude these constructs from further experiments. Instead we initiated starter cultures for the following plasmid combinations: 6+9, 6+10, 6+11, 8+9, 8+10, and 8+11. Each culture was transferred to a 50 mL Falcon tube containing LB broth supplemented with spectinomycin and ampicillin.

**25/09**

Following our experimental procedures, we incubated cells containing constructs 6+9, 6+10, 6+11, 7+9, 7+10, and 7+11 for 6 hours with varying concentrations of hydrogen peroxide. For these experiments we prepared 5 concentrations for each plasmid construct: 0mM, 5mM, 10mM, 20mM and 30mM. After 6 hours, we measured optical density (OD) at 600nm and fluorescence with excitation at 570nm and emission at 610nm to assess the biosensor response to oxidative stress and the resilience of our cells.

**26/09**

Following our initial incubation, we realized that we had omitted ampicillin when preparing the cells which compromised selection for the catalase plasmids. To address this, we repeated the incubation with hydrogen peroxide, this time allowing the cells to incubate overnight in a shaking incubator.

**27/09**

Remeasured absorbance and fluorescence for all cultures. These results showed improved responses compared to the previous tests, though the cells still struggled to survive even the lowest hydrogen peroxide concentrations. In light of this, we decided to lower both the concentrations and the incubation times for subsequent experiments.

**30/09**

ncubated cells for the third time with hydrogen peroxide, this time using 0mM, 2.5mM, 5mM, 10mM and 20mM H2O2. Measurements of absorption and fluorescence were taken at 1h, 3h and 4.5h intervals to evaluate the time-dependent effects of hydrogen peroxide on cell viability and biosensor performance.

**1/10**

We found that our results still did not adequately demonstrate biosensor accuracy or cell tolerance to oxidative stress. Instead, we decided to follow the alternative approach detailed in our experimental procedures to incubate cells with hydrogen peroxide, aiming to optimize cell growth for better survival and more reliable biosensor readings.

## References

## References

Bradley, R. W. An easy-to-use CRISPRi plasmid tool for inducible knockdown in E. coli. Biotechnol. Rep. 32, e00680 (2021).

### Sponsors

![Sponsor 1](https://static.igem.wiki/teams/5423/logo-anu-black.svg)
![Sponsor 2](https://static.igem.wiki/teams/5423/bpa-header.svg)
![Sponsor 3](https://static.igem.wiki/teams/5423/arc-logo.png)
![Sponsor 3](https://static.igem.wiki/teams/5423/igem.png)

### Contact Us

Email: anubioengineering@gmail.com

Phone: (+61) 434-389-738

Canberra, ACT, 2601, ANU

### Quick Links

[Contribution](https://2024.igem.wiki/anu/contribution) |
[Description](https://2024.igem.wiki/anu/description) |
[Engineering](https://2024.igem.wiki/anu/engineering) |
[Experiments](https://2024.igem.wiki/anu/experiments)

![Loading...](https://static.igem.wiki/teams/5423/untitled-design-2.gif)

[![ANU iGEM Logo](https://static.igem.wiki/teams/5423/anu-igem-logo-v3-1.png)](index.html)

Project

* [Contribution](https://2024.igem.wiki/anu/contribution)
* [Description](https://2024.igem.wiki/anu/description)
* [Engineering](https://2024.igem.wiki/anu/engineering)
* [Experiments](https://2024.igem.wiki/anu/experiments)
* [Notebook](https://2024.igem.wiki/anu/notebook)
* [Results](https://2024.igem.wiki/anu/results)

[Safety](https://2024.igem.wiki/anu/safety)
[Human Practices](https://2024.igem.wiki/anu/human-practices)

Team

* [Members](https://2024.igem.wiki/anu/team)
* [Attributions](https://2024.igem.wiki/anu/attributions)

Awards

* [Sustainable](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sustainable%27%29%20%7D%7D)
* [Education](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27education%27%29%20%7D%7D)

**Engineering**

![](https://static.igem.wiki/teams/5423/boo.png)

[ROS-inducible catalase](#v-pills-home)
[CRISPRi](#v-pills-profile)

## Engineering the ROS-inducible catalase system

### Design and Build

The engineering of our ROS-inducible catalase system began with a comprehensive design and construction phase. A critical initial step in designing our system was selecting candidate catalase genes for testing, which would enable optimisation of our system’s ROS-quenching abilities. We chose our candidates based on the following criteria:

* KatG from Escherichia coli served as our benchmark, as our testing was conducted in E. coli. We thought its established functionality and compatibility would provide a reliable reference point for evaluating the performance of other candidates.
* KatG from Mycobacterium tuberculosis, selected for its well-studied properties and effectiveness in neutralizing ROS [1].
* KatG from Bacillus, chosen as a wildcard candidate given previous reports of excellent ROS neutralising abilities [2].

After selecting our catalase genes, we had to slightly modify our KatG from E. coli plasmid to prevent recognition and targeting by our CRISPR system.

To facilitate the construction process, we employed Golden Gate Cloning, which allowed for the easy inclusion and swapping of genetic parts, streamlining the assembly of our plasmids and constructs. We utilised this cloning method to build cell lines containing catalase plasmids that are inducible by rhamnose, enabling us to switch the expression of these catlases on and off as needed.

In addition to constructing the catalase systems, we also decided to design and build biosensors to measure catalase effectiveness and evaluate suitable ROS-sensitive promoters. Using Golden Gate Cloning, we transformed plasmids containing different ROS sensitive promoters (Trxcp, SoxS and KatG) into E. coli. A second level of golden gate cloning enabled us to incorporate either oxyr or soxr in the ROS-sensitive plasmids, facilitating the construction of a ROS inducible system with RFP expression regulated by our promoters. This design allowed us to use fluorescence as a readout of promoter inducibility, enabling us to systematically evaluate which promoter would be most effective for our system.

### Test

After selecting our candidates, the next step in our engineering cycle was to identify the most effective and suitable catalase and promoter for our system. To optimize our approach, we first aimed to measure these components individually, before combining them into a single cohesive system.

After completing the transformations of our level 2 Golden Gate reactions, we observed that the induction of the TrxCP promoter was insufficiently linked to ROS levels. The plasmid-containing plates exhibited significant red fluorescence even in the absence of added ROS, suggesting that the promoter was leaky. Because of this, we deemed the Trxcp promoter unsuitable for our system and particularly inappropriate for future applications in a marine environment.

Consequently, we focused our further experiments only on the remaining promoters SoxS and KatG. Unfortunately we encountered challenges during the testing phase; our attempts to follow established protocols and hydrogen peroxide concentrations from existing literature [1,2] did not yield favorable results, as our cells struggled to survive even low concentrations of ROS.

To address this, in our second iteration of testing we drew inspiration from the methods of the HKUST 2022 iGEM team, Fisherly. They recommended incubating the cells during their log phase rather than stationary phase, significantly enhancing the cells’ resilience and our ability to measure promoter activity. This second cycle yielded more promising outcomes: the cells demonstrated improved tolerance to high concentrations of hydrogen peroxide, and the promoters effectively regulated the expression of fluorophores in response to ROS.

### Learn

Through these experiments, we identified the most optimal combination of promoters and catalases for our ROS-inducible system. We found that the SoxS promoter responded most effectively to ROS levels, demonstrating a strong baseline expression and robust and consistent responses to increasing concentrations of hydrogen peroxide. Additionally, cell survival was highest in the SoxS/KatG E. coli constructs, making this combination the most promising candidate for further engineering.

Moving forward, our focus will be on integrating these elements into a single engineered system. This integration will allow us to create a robust, responsive catalase system capable of efficiently mitigating ROS concentrations in a controlled manner. Once established, we can then test this system in other organisms, with the aim of implementing it in the coral microbiome.

## References

1. Manca, C., Paul, S., Barry, C. E., Freedman, V. H. & Kaplan, G. Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro. Infect. Immun. 67, 74 (1999).
2. Jia, X., Chen, J., Lin, C. & Lin, X. Cloning, Expression, and Characterization of a Novel Thermophilic Monofunctional Catalase from Geobacillus sp. CHB1. Biomed Res. Int. 2016, (2016).

## Engineering the CRISPRi system

Earlier studies have found that bacteria favour the production of native genes over analogues of these genes inserted via plasmids. For example, our expression bacteria (Escherichia coli DH10B) would favour its native KatG over the Mycobacterium tuberculosis KatG. To optimise the production of such proteins, we looked for analogues of the potential catalases, where we noted that only M. tuberculosis KatG has an E. coli analogue. We then attempted to use a CRISPR interference (CRISPRi) system to knockdown native KatG production, hoping to increase the rate of M. tuberculosis KatG production.

### Design and Build

We chose a CRISPRi system from Bradley1 for a few reasons. Firstly their plasmid utilised a p15A backbone, which would be compatible with the plasmids used for catalase production. Secondly, it uses a chloramphenicol antibiotic resistance (ABR) gene which is different from the ABR genes used by the catalase team. Finally, this system is run on one plasmid where both the dCas9 and gRNA are present on the same plasmid (unlike many other CRISPRi systems that use a seperate plasmid for the dCas9 and gRNA).

### Test

When attempting to replicate an experiment by Bradley1 to confirm that the system works as expected. Bradley1 showed that their system resulted in a 40-fold repression of mScarlet 3 (mScar3), however we were unable to replicate this. To optimise this system to our needs, we increased the concentration of L-arabinose (Ara)- which induces gRNA production- resulting in a slight increase in repression. To further optimise the system, we attempted to alter the concentration of anhydrotetracycline (aTc)- which induces the dCas9- however we were unable to reach a 40-fold repression. We noticed that this plasmid contained a H840D in the dCas9 sequence (where other plasmids generally use a H840A to deactivate Cas9). We attempted to revert this using site-directed mutagenesis however this was unsuccessful. Finally, we performed sanger sequencing on the gRNA ensuring that the guide sequence was correct.

### Learn

Through our engineering process we have produced two new plasmids as part of a CRISPRi system. However, our testing revealed that this system does not work. We were unable to pinpoint the exact reason, but it it is possible that the dCas9 is faulty. ChopChop2 was used to determine the optimal gRNA sequence, thus it is highly likely that the gRNA sequence is on-target. In future iterations we plan to use a different CRISPRi system with a similar gRNA sequence.

## References

1. Bradley, R. W. An easy-to-use CRISPRi plasmid tool for inducible knockdown in E. coli. Biotechnol. Rep. 32, e00680 (2021).
2. Labun, K. et al. CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res. 47, W171–W174 (2019).

### Sponsors

![Sponsor 1](https://static.igem.wiki/teams/5423/logo-anu-black.svg)
![Sponsor 2](https://static.igem.wiki/teams/5423/bpa-header.svg)
![Sponsor 3](https://static.igem.wiki/teams/5423/arc-logo.png)
![Sponsor 3](https://static.igem.wiki/teams/5423/igem.png)

### Contact Us

Email: anubioengineering@gmail.com

Phone: (+61) 434-389-738

Canberra, ACT, 2601, ANU

### Quick Links

[Contribution](https://2024.igem.wiki/anu/contribution) |
[Description](https://2024.igem.wiki/anu/description) |
[Engineering](https://2024.igem.wiki/anu/engineering) |
[Experiments](https://2024.igem.wiki/anu/experiments)

![Loading...](https://static.igem.wiki/teams/5423/untitled-design-2.gif)

[![ANU iGEM Logo](https://static.igem.wiki/teams/5423/anu-igem-logo-v3-1.png)](index.html)

Project

* [Contribution](https://2024.igem.wiki/anu/contribution)
* [Description](https://2024.igem.wiki/anu/description)
* [Engineering](https://2024.igem.wiki/anu/engineering)
* [Experiments](https://2024.igem.wiki/anu/experiments)
* [Notebook](https://2024.igem.wiki/anu/notebook)
* [Results](https://2024.igem.wiki/anu/results)

[Safety](https://2024.igem.wiki/anu/safety)
[Human Practices](https://2024.igem.wiki/anu/human-practices)

Team

* [Members](https://2024.igem.wiki/anu/team)
* [Attributions](https://2024.igem.wiki/anu/attributions)

Awards

* [Sustainable](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sustainable%27%29%20%7D%7D)
* [Education](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27education%27%29%20%7D%7D)

**Experiments**

![](https://static.igem.wiki/teams/5423/book.png)

* [ROS-inducible Catalase](#ros-inducible-catalase)
* + [1.Overview](#overview)
  + [2.Plasmid Construction](#plasmid-construction)
  + [3.H2O2 Experiments](#hydrogen-peroxide-experiments)
* [CRISPRi Experiments](#crispri)
* + [1.Plasmid Construction](#crispri-plasmid-construction)
  + [2.Functionality Testing](#crispri-functionality)

## ROS-inducible catalase experiments

### Experimental Overview

Our primary experiments focus on the engineering of a ROS-inducible catalase system designed to effectively neutralise reactive oxygen species within the coral microbiome and thereby protecting them from bleaching. This system is intended to be optimized for both control - selecting the best promoter that elicits the most effective response to ROS - and neutralization - identifying the most effective catalase for this application.

To facilitate the assembly and optimization of our system, we utilised Golden Gate cloning, allowing us to efficiently combine various components as needed. This modular approach enhanced our ability to iteratively refine our system.

For our promoter selection, we chose:

* **OxyR/KatG:** the native promoter for KatG in E. coli.
* **OxyR/TrxCp:** another E. coli promoter known for its ROS sensitivity.
* **SoxR/SoxS:** a superoxide E. coli promoter that may facilitate multi-ROS activity.

For the catalases, we selected:

* **KatG** from E. coli.
* **KatG** from Mycobacterium tuberculosis (strain H37Rv)
* **Kat** from Geobacillus sp. CHB1.

To validate our system, initial laboratory testing was conducted in E. coli, which provided a controlled environment to assess the effectiveness of these components before progressing to more complex systems in the coral microbiome.

### Plasmid construction

Golden gate reactions

All the reagents and gBlocks used for the construction of each clone are detailed in the Cloning Guide. Constructs 1-5 were used to make 6-8, which served as biosensors. In these constructs, RFP was regulated by our ROS-sensitive promoters to measure their efficacy under increasing concentrations of hydrogen peroxide. Constructs 9-11 were designed to express our 3 catalase genes, using a rhamnose promoter which acted as an ON/OFF switch to provide simple negative controls.

Procedure:

* Prepared the necessary reagents (see cloning guide) and gBlocks for golden gate cloning
* Combined DNA fragments and reaction mixture in a PCR tube.
* Incubated the reaction mixture in the thermocycler, cycling between digestion and ligation.

Transformations

1. Thawed 100uL E coli DL21 cells and glycerol on ice.
2. Thawed 100uL E coli DL21 cells and glycerol on ice.
3. Combined 100uL cells and 1uL golden gate reaction mixture in a cuvette for each construct
4. Prepared 900uL of Super Optimal Broth for each reaction
5. For each construct, applied an electric pulse and immediately added the mixture to the SOB.
6. Recovered in the SOB for 1 hour at 37°C.
7. Spread 100uL of transformed cells onto plates containing selective media (see Notebook) and incubated overnight at 37 degrees.

Picking colonies

1. Selected individual colonies from the transformation plates using a sterile pipette tip.
2. Transferred each colony into LB broth with selective media (see Notebook).
3. Incubated the cultures overnight at 37°C on a shaking incubator.

Miniprep

1. Pelleted the cells by centrifugation at 13,000 rpm for 1 minute.
2. Discarded the supernatant and resuspended the cell pellet in 250 µL of resuspension buffer.
3. Added 250 µL of lysis buffer to the resuspended cells and mixed gently by inversion.
4. Added 350 µL of neutralisation buffer and mixed gently by inversion.
5. Centrifuged the mixture for 10 minutes at 13,000 rpm to pellet cell debris.
6. Transferred the supernatant to a spin column and centrifuged for 1 minute.
7. Washed the column with 500 µL of wash buffer and centrifuged for 1 minute.
8. Repeated the wash step with another 500 µL of wash buffer, followed by centrifugation.
9. Eluted the plasmid DNA by adding 50 µL of elution buffer to the center of the column and centrifuged for 1 minute.
10. Measured the concentration of plasmid DNA using Nanodrop

Digest

1. Added 4.5 uL of each plasmid, 1 uL cutsmart buffer, 4 uL H2O and 0.5 uL of NdeI restriction enzymes into microtubes.
2. Incubated in a thermocycler overnight.

### Hydrogen peroxide experiments

Incubation of cells with hydrogen peroxide (constructs 6+9, 6+10, 6+11, 8+9, 8+10, 8+11)

1. For each construct, transferred 300uL starter culture and 20mL LB with spec and carb to new Falcon tubes.
2. Measured the optical density to ensure it was in the range of 0.05-0.08.
3. Prepared hydrogen peroxide solutions in Round-Bottom Polypropylene Test Tubes to achieve final concentrations of 0mM, 5mM, 10mM, 20mM and 30mM, given a final volume of 2mL and from a 1M stock. For each concentration, there should be one tube for every construct, labelled with both the plasmids (eg 6+9) and the H2O2 concentration (eg 0mM).
4. For the negative controls, added 2mL of diluted culture without rhamnose to corresponding tubes (constructs 6+9 and 8+9)
5. Added 500uL 20% L-rhamnose to each 20mL dilution to induce catalase plasmid expression.
6. Transferred 2mL of each culture to corresponding test tubes.
7. Incubated at 37 degrees on a shaking incubator (exact times described in Notebook)

Measuring OD and fluorescence

1. Added 100uL of each culture incubated with hydrogen peroxide to a 96-well plate.
2. Measured absorbance at 600 nm using Infinite M Plex plate reader.
3. Measured fluorescence with excitation at 570 nm and emission at 610 nm.

Incubation of cells with hydrogen peroxide

1. Transferred 100uL of each starter culture and 5mL LB with spec and carb to new 50mL Falcon tubes
2. Incubated on a shaking incubator at 37 degrees for 2 hours, until the cultures reached 0.7-1 OD600. At this stage, the cells should be in their log phase.
3. Back-diluted the cultures to an OD600 of 0.2 by diluting them in LB, bringing the total volume to 7.5 mL in new 15 mL Falcon tubes.
4. Prepared hydrogen peroxide solutions in 1.5mL Eppendorf Tubes to achieve final concentrations of 0mM, 0.25mM, 0.5mM, 1mM and 2.5mM and 5mM, given a final volume of 1mL. For each concentration, we used one tube for every construct.
5. Added 1 mL of diluted culture (without L-rhamnose) to the corresponding test tubes for constructs 6+9 and 8+9, to act as negative controls.
6. For all cultures, including constructs 6+9 and 8+9, added 190 µL of 20% L-rhamnose to each 7.5 mL dilution to induce catalase plasmid expression.
7. Transferred 1mL of each culture (including 6+9 and 8+9) to corresponding remaining test tubes.
8. Incubated at 37 degrees on a shaking incubator for 3 hours

Measuring OD and fluorescence

1. Added each culture incubated with hydrogen peroxide to a 96-well plate.
2. Measured absorbance at 600 nm using Infinite M Plex plate reader.
3. Measured fluorescence with excitation at 570 nm and emission at 610 nm.

## CRISPRi engineering

### Plasmid Construction

Oligo Annealing

1. Dilute primers to 100 uM using H2O.
   * Katg gRNA forward strand
   * Katg gRNA reverse strand
   * mScarlet3 gRNA forward strand
   * mScarlet3 gRNA reverse strand
2. Add 2uL of each partner strand to 14 uL of H2O and 2 uL of 10x annealing buffer
3. Anneal the strands: 5 minutes at 94 °C, then 70 x -1 °C per minute until the reaction vessel reaches 24°C

BsaI Reaction

1. Dilute the annealed samples by 100x
2. Add 1 uL Ligase Buffer, 7 uL H2O, 0.5 uL diluted annealed sample, 0.5 uL pdCas9, 0.5 uL Bsa1 and 0.5 uL ligase to give sixfold excess of guide RNA
3. Cycle the strands: 50 x 5 minutes at 37 °C, 5 minutes at 16 °C, then 10 minutes at 37 °C, followed by 20 minutes at 80 °C

pdCas9 Cloning

1. (strain) E. coli were transformed with the plasmids via electrocution. The cells were shocked in the presence of the plasmid, then diluted into LB to allow them to recover.
2. Selection plates were made with LB agar and chloramphenicol
3. The transformed cells were pelleted and plated onto the selection plates.
4. Plates were incubated overnight at 37 °C
5. Colonies were selected and grown overnight in LB broth with chloramphenicol
6. Plasmids were isolated by Isolate II Plasmid Mini Kit
7. DNA concertation was read by NanoDrop
8. The identity of the plasmids was confirmed via an enzyme digest, and sanger sequencing

1. (strain) E. coli were transformed with the following plasmid combinations as per the procedure in step 7
   * Combination 1 – Purified Katg pdCas9 Plasmid
   * Combination 2 – Purified mScarlet3 pdCas9 Plasmid + pET16 Rhamnose mScarlet3
   * Combination 3 – Purified mScarlet3 pdCas9 Plasmid + pCDF J23100 (constitutive) mScarlet3
2. Selection plates were made with chloramphenicol (to screen for combo 1), chloramphenicol + carbenicillin (to screen for Combo 2), and chloramphenicol + spectinomycin (to screen for combo 3)
3. The cells were pelleted and plated onto their respective plates.
4. 3-4 Colonies were selected from each plate and grown overnight in LB broth with their corresponding selection antibiotics.

### CRISPRi Functionality Testing

To validate the function of the CRISPRi system, we replicated an experiment performed by Bradley (2021).

1. Combination 2 and 3 bacteria (from infusion transformation step) were used.
2. An example set up is shown below:

| Conditions | A | B | C | D | E |
| --- | --- | --- | --- | --- | --- |
| Rhamnose | - | + | + | + | + |
| Arabinose | - | - | + | - | + |
| anhydrotetracycline | - | - | - | + | + |

Where + indicates the addition of an inducer.

1. Each combination was grown in 3 mL lysogeny broth (LB)
2. Chloramphenicol stock of 33 mg/mL was added to LB to result in a final concentration of 33 µg/mL
3. Anhydrotetracycline stock of 100 µg/mL was added to the required conditions in table x. to result in a final concentration of 100 ng/mL
4. Rhamnose stock of 20% (m/v) was added to the required conditions in table above. to result in a final concentration of 0.2% (m/v)
5. Arabinose was added to result in a final concentration of 1.2 mM
6. A “high arabinose” concentration was also used of 26.6 mM (as a separate condition)
7. Following this, cultures were grown overnight at 37°C
8. On the next day, take 150 µL of overnight cultures and place into clear 96 well plate (Costar 96 U Bottom Transparent Polystyrene).
9. Absorbance at 600nm (OD600) was measured using Infinite M Plex plate reader.
10. 100 µL of the solution from the clear 96 well plate is transferred to a black 96 well plate (Costar 96 Flat Bottom Black Polystyrene).
11. mScarlet 3 fluorescence was measured using an excitation wavelength of 560nm and an emission wavelength of 620nm.

KatG growth test

1. 3-4 colonies were selected from each of the purified pdCas9 plates grown overnight in selective LB broth.
2. 600 uL of incubated sample was added to 60 mL of new LB broth with 300 uL L-arabinose
3. The samples were incubated for 2.5 hours, then split into two. One replicate of each plasmid was given 30 uL aTc, and optical density at 600nm (OD600) was measured:
4. OD600 was measured at 30 minute intervals for 1 hour.
5. 0.1mM H2O2 was added to the samples
6. OD600 was recorded after 30 minutes of growth

## References

Bradley, R. W. An easy-to-use CRISPRi plasmid tool for inducible knockdown in E. coli. Biotechnol. Rep. 32, e00680 (2021).

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* [ROS-inducible Catalase](#home)
* [CRISPRi](#profile)

## ROS-inducible Catalase Results

Our investigation of the performance of the KatG, SoxS and TrxCP promoters revealed notable differences in their responses to varying concentrations of hydrogen peroxide (H2O2) (Figure 1). The native E. coli KatG promoter showed the lowest relative expression of RFP among the promoters tested and exhibited only minimal increases in expression as H2O2 levels rose. Due to its low baseline activity and weak inducibility, we decided that the KatG promoter is unsuitable for our ROS-inducible system, and is not the most promising candidate moving forwards.

![](https://static.igem.wiki/teams/5423/biosensor-fluorescence.png)

**Figure 1:** Response of our ROS-sensitive biosensors to increasing hydrogen peroxide (H2O2) concentrations. The fluorescence of RFP, normalized to cell OD600, is shown under the control of three different ROS-sensitive promoters.

In contrast, both the SoxS and TrxCP promoters showed similar expression patterns with fluorescence peaking at 2500uM H2O2 before declining at 5000uM. Given that both these promoters are ROS inducible this decrease is unexpected, as we would typically anticipate further increases in fluorescence at higher concentrations. Additionally, we noted significant red fluorescence on plates containing the TrxCP plasmid even in the absence of added ROS. This observation indicates a leakiness of the TrxCP system over time, rendering it unsuitable for our aim to implement our system in the coral microbiome. Instead, we recommend retesting the SoxS promoter to confirm the decline in fluorescence at 5000uM. Despite this decrease the SoxS promoter still exhibited higher expression than KatG at these concentrations, positioning it as the most promising candidate for further testing.

![](https://static.igem.wiki/teams/5423/catalase-fluorescence-option1.png)

**Figure 2:** Response of our ROS-sensitive biosensors in combination with catalase plasmids to increasing H2O2 concentrations. The fluorescence of RFP, normalised to cell OD600, is shown for each construct.

These findings are further supported by the fluorescence analysis of our three ROS-sensitive biosensors in conjunction with catalase plasmids exposed to varying concentrations of H2O2. Due to the leakiness of the TrxCP promoter, we decided to exclude it from these analyses and instead focused solely on the KatG and SoxS promoters. The KatG promoter exhibited a weak correlation between fluorescence and H2O2 concentration, reaffirming its unsuitability for a ROS induced system. In contrast, the SoxS promoter showed strong and consistent induction by ROS, with a clear activation threshold at 2500 µM across all four constructs. This highlights its suitability for ROS detection and solidifies it as the most promising candidate for our system.

As expected, cell survival analysis revealed that all cell lines displayed higher optical densities (OD) at lower H2O2 concentrations, reflecting enhanced viability under less toxic conditions (Figure 3). Interestingly the cells containing the SoxS promoter exhibited significantly better survival rates than those with the KatG promoter across all tested concentrations. In fact, cells expressing the KatG promoter alongside catalase plasmids displayed survival rates comparable to the biosensors alone when exposed to hydrogen peroxide. This observation suggests that the catalase plasmids in these cells do not significantly enhance cell survival. Instead, it suggests a potential detrimental interaction between the KatG promoter and all three of the catalase plasmids, which may hinder their protective effects against oxidative stress.

**(a)**
![](https://static.igem.wiki/teams/5423/combined-od-option1.png)

**(b)**
![](https://static.igem.wiki/teams/5423/catalase-od-option1.png)

**Figure 3:** Effect of varying hydrogen peroxide (H2O2) concentrations on cell survival of E. coli cell lines expressing catalase plasmids. **(a)** The colored constructs represent cell lines containing one of our three catalase genes paired with a second plasmid expressing RFP under the control of the SoxS (red) or KatG (blue) promoter, with catalase expression induced by rhamnose. Constructs marked as negative were not induced. The catalase expression is inducible by rhamnose, and negatives are not induced while positives are. Black lines indicate the survival of control cell lines that do not express any catalase plasmids. **(b)** Colored bars represent OD600 of cell lines containing one of our three catalase genes paired with a second plasmid expressing RFP under the control of the SoxS or KatG promoter, with catalase expression induced by rhamnose. The results are grouped by construct.

Among the catalases, the native KatG from E. coli demonstrated superior survival with minimal changes in OD as ROS levels increased when combined with the SoxS promoter. However, when paired with the KatG promoter, these plasmids demonstrated the poorest performance, indicating a potential detrimental interaction. This may be attributed to the introduction of both the native E. coli KatG and its corresponding promoter into E. coli, potentially leading to unexpected regulatory interactions. Future optimisation efforts should focus on determining whether these effects are specific to the tested organism (E. Coli) or whether a similar pattern is seen in other organisms.

Furthermore, the negative controls (lacking rhamnose induction) showed performance similar to that of the rhamnose-induced plasmids. This outcome contradicts our expectation that plasmid catalase expression would not occur without induction, and therefore these cells should exhibit decreased viability. This unexpected finding raises the possibility that even basal levels of catalase expression in E. coli may be sufficient to confer cell resilience under these conditions, warranting further investigation. Future experiments should explore a wider range of H2O2 concentrations to determine the overall resilience, and incoporate CRiSPR knockdown of the native catalase to better understand its role in these cells’ survival.

Overall, our results suggest that the most promising combination of promoter and catalase is the SoxS promoter with KatG from E. coli. However, experiments should first validate the performance of each of these parts individually in other organisms. After validation, these plasmids can be integrated into a single system, allowing the catalase to be regulated by the ROS-inducible promoter and eventually incorporated into the coral microbiome.

## CRISPRi Results

Despite confirmation of successful gRNA primer insertion, via enzyme digest (Fig.1) and sanger sequencing, we were unable to replicate the levels of repression observed in the source paper1, with initial testing showing less than 20% percent reduction (Fig.2).

![](https://static.igem.wiki/teams/5423/enzyme-digest.png)

**Figure 1:** Gel electrophoresis following BamHI and SacII restriction enzyme digest of gRNA inserted plasmids. The three lanes on the left is a computational prediction of the gel electrophoresis, while the four wells on the right are the experimental results. MW refers to the DNA ladder. Well 1 of the computationally predicted gel represents the BsaI and SacII enzyme digest of the initial, unreacted, plasmid, whilst well 2 represents the digest of the plasmid following gRNA insertion. Computational well 2 represents the digest of the plasmid following gRNA insertion. Experimental well 1 is the plasmid backbone without an inserted gRNA. Experimental well 2 is the plasmid backbone with the KatG gRNA inserted. Experimental well 3 is the plasmid backbone with the mScarlet3 gRNA inserted.

![](https://static.igem.wiki/teams/5423/highara-hist-final.png)

**Figure 2:** Inducing production of dCas9, gRNA and mScarlet3 to measure CRISPRi activity. + indicates the presence of the inducer, while - indicates absence. Rhamnose induces mScarlet3 production. Anhydrotetracycline induces dCas9. L-arabinose induces gRNA. ++ indicates that the L-arabinose concentration was 26.6 mM. Constitutive in the rhamnose row indicates that a constitutive promoter was used and thus rhamnose was not added. Fluorescence was normalized to mScarlet3 induction only (condition C).

Following this, we attempted to optimize for [anhydrotetracycline] and [L-arabinose], but this was also unsuccessful, with changing concentrations resulting in decreased activity relative to the source paper1conditions (Fig.3). As such we have decided to discontinue the CRISPRi element of the project. Perhaps CRISPRi system would have found more success against the genomic E. coli KatG, due to the lower transcription rate (relative to the mScarlet3 plasmid), we were unable to test this with rigor. Furthermore, current scientific literature suggests that CRISPRi systems work better when the gRNA targets a promoter. As such, in the future it would be beneficial to design a plasmid system in which the gRNA targets the KatG/mScarlet3 promoter. However, this would require the use of a different CRISPR system, as the DH10B E. coli KatG promoter does not contain the dCas9 protospacer adjacent motif.

![](https://static.igem.wiki/teams/5423/rha-atc-altered-hist-final.png)

**Figure 3:** Varying anhydrotetracycline and rhamnose concentration. Rhamnose induces mScarlet3 and anhydrotetracycline induces dCas9. Results are normalized to concentrations used by the source paper1.

### References

1. Bradley, R. W. An easy-to-use CRISPRi plasmid tool for inducible knockdown in E. coli. Biotechnol. Rep. 32, e00680 (2021).

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### Contact Us

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Phone: (+61) 434-389-738

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