iGEM 2022 — Team umanitoba

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{% block title %}Notebook{% endblock %}
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# Note Book

##### April - May (Planing, Brainstorming)

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- Brainstorming & Research

- Met with Brendin Spearing (Boat inspector with Aquatic Invasive Species)

- Met with Candace Parks (Aquativ Invasive species expert)

- Design and order DNA for sensors

- Begin *in sillico* work for toxic proteins

##### June (Cloning)

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(Week 1) Insert of EGFP with BamHI and HindIII cut sites are isolated. Plasmid database/inventory is created.

(Week 2) Synthesized constructs are confirmed with the correct size and entered into the plasmid inventory. Initial cloning experiments yield few colonies, but isolated products do not have the correct fragment size.

(Week 3) Cloning experiments yield few colonies, and most isolated products do not have the correct fragment size. One experiment gave a product with the T7 promoter with correct size fragments after digestion.

(Week 4) Cloning protocols are not reproducible as further attempts to clone yield no colony. Time to try a different assembly strategy.

##### July (Still Cloning)

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(Week 1) Successful cloning of EGFP into T7 promoter construct (pUCIDT plasmid). Obtain digested pSB1C3 backbone for further cloning purposes.

(Week 2) Expression of EGFP using T7\_EGFP in pUCIDT with IPTG as inducer. Detection of expression using SDS-PAGE and fluorescent spectroscopy.

(Week 3) T7\_EGFP construct is successfully cloned into pSB1C3 backbone. PCR gives a poor quantity of inserts and backbone due to non-specific binding. Both LB and M9 media are suitable for EGFP expression with LB being slightly better (higher fluorescent/OD600). M9/Tris medium is not suitable for growing cells as OD600of cells is very low in comparison the to other two media.

(Week 4) Key outcomes: Gel extraction gives very low-quality and low-yield DNA products for cloning purposes. Expression of EGFP is detected in nitrate and phosphate sensing cells. Response curves in response to concentrations of nitrate (0 - 500 μM) and phosphate (0 – 500 μM) are constructed. No expression is detected in pSB1C3\_T7\_EGFP.

FitD sequence and aerolysin sequence are finalized and ordered.

##### August (Chracterize Sensors + More Trouble shooting & Cloning)

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Optimization is still required for cloning experiments. By the end of the week, good quality gel extracted DNA samples of nitrate-sensor-EGFP insert, phosphate-sensor-EGFP insert, and pSB1C3 backbone are obtained for setting up the ligation reaction. Expression of EGFP in response to phosphate and nitrate concentration is reproduced and thus, further proves that the designed construct works!

DNA fragments required for Gibson Assembly are obtained by using new primers. Nitrate sensor\_EGFP and phosphate sensor\_EGFP are successfully cloned into pSB1C3. Expression results are reproducible.

T7\_EGFP is successfully cloned into pET28b. Toxin-containing plasmids are documented and prepared for expression. No EGFP expression is detected for nitrate and phosphate sensing bacteria with pSB1C3 backbone.

Nitrate sensor\_EGFP is successfully cloned into pET28b backbone the quality of DNA samples for cloning is poor. No expression of EGFP is detected for pH and oxygen sensors.

##### September (Toxins & Protein Work)

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T7\_Act is successfully cloned in a pET28b backbone. No expression of EGFP is detected for pH and oxygen sensors. It is unclear whether EGFP is expressed using a nitrate sensor in pET28b backbone.

T7\_FitD is successfully cloned into pET28b backbone. EGFP expression is best for nitrate sensors in pUCIDT (very high copy number). Some expression is observed in pET28b (low copy number) and negligible expression is observed in pSB1C3 (high copy number).

Detection of FitD by SDS-PAGE analysis proves difficult. Aerolysin purified by IMAC chromatograph

Toxicity assays completed by UNILausanne

Finished RNA aptamers assay for Lethbridge High School

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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# Engineering

##### Engineering Principles

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Engineering principles were used throughout the ZebraZap Project by adopting the **D**esing-**B**uild-**T**est-**L**earn cycle in the overall project design. Furthermore, a strong emphasis on predictive design allowed us to successfully design an active variant of FitD that is nearly a third the length of the original. Engineering is a diverse discipline, therefore there is lots of variety when it comes to engineering principles. In addition to modelling, the engineering principles most used throughout ZebraZap are highlighted below.

###### Decomposition

The act of breaking up a large complex system into a number of simpler subsystems. The very first thing that we did in this project was split the problem of controlled expression of a toxin into the promoters (sensors) and toxins (actuators).

###### Decoupling

Also referred to as functional isolation or modular thinking. Assume that the different abstract pieces that make up your project don’t interact with each other, they are totally interchangeable. A natural progression from decompostion. Test and debug all the systems independently and then integrate them at the end.

###### Parallelization

Once you adopt the ideas of decomposition and decoupling there is no need to wait for one part to be done before working on the others. This allows projects to be completed efficiently.

###### Standardization

Standardized parts from the registry were used extensively in ZebraZap. This allowed us more time to focus on designing our unique parts carefully and minimizing unwanted surprises.

---

##### Note

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In addition to the large-scale design of the project the individual elements such as aerolysin and FitD, were realized by adherence to engineering principles. The page will be dedicated to the promoter system as a clear example of engineering biology.

##### Design (I)

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After three rounds of consultation B.Spearin and C. Parks, experts from the aquatic invasive species center, we had an idea for what we considered the ideal bio-control device. It would exist passively in the environment until it detected Zebra Mussels at which point it would deliver a highly specific and non-bioaccumulating toxin and then turn off.

For the effectors, we decided on the set of proteins FitD and aerolysin. Since the effectors were a protein they made promoters a logical choice for the sensors. This meant the entire device could be assembled in the same plasmid.

The next step was to consider how we should go about detecting the zebra mussels. This took the form of a literature search for all the hallmarks of zebra mussels’ infestation. The most consistent observation was that water clarity significantly increased as a result of the various filter feeders (Secchi transparency). We considered a number of light-sensitive promoter systems but eventually ruled them out. We reasoned that this may not be practical and factors like water depth, shade and the day-night cycle would cause too much interference. Chlorophyll levels also drop significantly, and this could still be a viable detection method. We considered a genetic approach, but the genomes are not that well annotated. The complete genome only is published for the first time in 20211.

So, we ruled out chlorophyll, water clarity and nucleic acids.

After searching through academic literature and government water quality reports we eventually found a reasonable consensus. The general observation seemed to be that as a result of the zebra mussel invasion there was a significant increase in nitrates and a decrease in phosphates.2-7We then chose the nitrate-induced BBa\_K216005 and the phosphate represses BBa\_K1682012.

Since we chose standard parts from the registry, this simplified the process of modelling using iBiomSym. The sensors seemed to have sufficient sensitivity at the concentrations we would need to work at. We also noted that the transition in the nitrate promoter seemed more abrupt than in the phosphate. We hypothesized that the nitrate promoter would allow for switch-like behaviour while the phosphate would be more gradual, like a dimmer.

![](https://static.igem.wiki/teams/4323/wiki/promoter-stuff/ibiosim-nitrate.png)

Figure 1. Nitrate promoter system simulated using iBioSym as outlined in the experimental methods.

![](https://static.igem.wiki/teams/4323/wiki/promoter-stuff/ibiosim-phosphate.png)

Figure 2. Phospate promoter system simulated using iBioSym as outlined in the experimental methods.

##### Build/Test/Learn (I)

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Initially, we had intended to order the genes by synthesis and use BioBrick assemble to design a modular construct. Despite attempting several different protocols ligation was very inefficient. The project was completed instead using Gibson assembly.

The sensors were validated by measuring the fluorescence over optical density. Our original idea was to immediately move the promoter into the iGEM standard pSBS1C3 plasmid. We wanted to get the system into the standard backbone as soon as possible for characterization. We performed the fluorescence measurement and were surprised to find that the results were very poor.

We were surprised to find that eGFP expression was so low. We hypothesized that the reason for the low gene expression is that the pSB1C3 is a high copy number plasmid and that switching to a low copy number plasmid pET28b would be better suited to protein expression. At the time we considered pSB1C3 and pUCIDT interchangeable, as they are both high copy number plasmids and did not think to test expression in pUCIDT.

##### DBTL (II)

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We moved our construct in its entirety into a pET28b plasmid.

Repeated the fluorescence titration experiment and the results were once again very poor.

We were surprised to find that eGFP expression was so low. We hypothesized that the reason for the low gene expression is that the pSB1C3 is a high copy number plasmid and that switching to a low copy number plasmid pET28b would be better suited to protein expression. At the time we considered pSB1C3 and pUCIDT interchangeable, as they are both high copy number plasmids and did not think to test expression in pUCIDT.

##### DBTL (III)

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We performed the fluorescence characterization with the pUCIDT vector, and recorded a good dose-response curve that could be used as a starting point to optimizing the nitrate and phospate systems.

![](https://static.igem.wiki/teams/4323/wiki/promoter-stuff/nitrate-in-different-plasmids.png)

Figure 3. Graph summarizing the testing that was done to get the nitrate promoter behaving as desired. The experiment was repeated with more presicion after wards.

![](https://static.igem.wiki/teams/4323/wiki/promoter-stuff/nitrate-hill-function.png)

Figure 4. The nitrate sensor BBa\_K216005 cloned into pUCIDT with expression measured by fluorescence. Fit to a four parameter Hill equation.

![](https://static.igem.wiki/teams/4323/wiki/promoter-stuff/phosphate-hill-function.png)

Figure 5. The phospate sensor BBa\_K1682012 cloned into pUCIDT with expression measured by fluorescence. Fit to a four parameter Hill equation.

##### DBTL (IV) ?

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At this point, we were ready to either clone in the toxic genes or alter the strength of the ribosome binding site to begin to more closely match nitrate levels in the actual environment. We learnt that all high-copy number plasmids are not interchangeable and that if you use modelling software the predictions may be more specific than you think. While this may not be the version of the DBTL we had envisioned, it allowed us to “debug” our oversight by proceeding in a systematic fashion.

We excitedly planned another meeting with an aquatic species expert and then had to make yet another course correction. We were informed that government agencies have been monitoring the chemical concentrations of Manitoban waters, and there are not any consistent changes to water chemistry that can be associated with zebra mussels.

So, we had to table our ideas for new nitrate sensors and choose a different target. Ultimately this changes the sensing from the external environment to the internal environment of the zebra mussel stomach. The stomach of the mussels is an anoxygenic acidic environment.

We considered three pH sensors from the registry, as well as the O2 sensor, BBa\_ K239005

BBa\_K3196028: pH 2-4

BBa\_K1231000: pH < 5.5

BBa\_K1231001: pH 5-7 (This was the most relevant pH range)

We had thought it would just be extremely acidic but some literature searching revealed otherwise. Stomach acid pH varies between organisms depending on how that organism is adapted to feeding.8 In humans, the stomach pH can be low as 1.5 while in chickens, the pH is around 3.7.8Scavengers and carnivores generally have a lower stomach acid pH while herbivores are higher. 8 For Zebra mussels, the stomach acid pH has fortunately been reported to be between 6.0-6.9 depending on the phase of digestion. 9.

Due to difficulties in testing, the oxygen and pH systems could not be made functional in time for the end of the iGEM 2022 season.

##### Engineering Succsess

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Detectors for a change in water chemistry, it had all seemed so neat and tidy on paper. Even though we used standardized parts it took many rounds of trial and error to get the promoters working. Had we used to pUCICT right away the project would have gone smoother, but we did not. Because we had a systematic approach we were able to catch the error and get the desired function.

##### References

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(1) McCartney, M. A.; Auch, B.; Kono, T.; Mallez, S.; Zhang, Y.; Obille, A.; Becker, A.; Abrahante, J. E.; Garbe, J.; Badalamenti, J. P.; Herman, A.; Mangelson, H.; Liachko, I.; Sullivan, S.; Sone, E. D.; Koren, S.; Silverstein, K. A. T.; Beckman, K. B.; Gohl, D. M. The Genome of the Zebra Mussel, Dreissena Polymorpha: A Resource for Comparative Genomics, Invasion Genetics, and Biocontrol. *G3 Genes, Genomes, Genet.* **2022**, 12 (2). <https://doi.org/10.1093/g3journal/jkab423>.

(2) Effects of recent zebra mussel invasion on water chemistry and phytoplankton production in a small Irish lake. Higgins et al.,[. **2008** http://www.aquaticinvasions.net/2008/AI\_2008\_3\_1\_Higgins\_etal.pdf](http://www.aquaticinvasions.net/2008/AI_2008_3_1_Higgins_etal.pdf)

(3) Retention of N and P by zebra mussels (Dreissena polymorpha Pallus) and its quantitative role in the nutrient budget of eutrophic Lake Ekoln, Sweden. *Biological Invasions* **2011**, <https://link-springer-com.uml.idm.oclc.org/article/10.1007/s10530-011-9950-9>

(4) Impact of Zebra Mussel Invasion on River Water Quality. Effler, S. W., Brooks, C. M., Whitehead, K., Wagner, B., Doerr, S. M., Perkins, M., Segfried, C.A., Walrath, L., Canale, R. P. **1996** <https://www.jstor.org/stable/25044708>

(5)Ecosystem-Level Effects of Zebra Mussels (Dreissena polymorpha): An Enclosure Experiment in Saginaw Bay, Lake Huron. 1995. Heath, R. T., Fahnenstiel, G.L., Gardner, W.S., Cavaletto, J.F., Hwang, S. *INternat. Assoc, Great Lakes* **1995** <https://datospdf.com/download/ecosystem-level-effects-of-zebra-mussels-dreissena-polymorpha-an-enclosure-experiment-in-saginaw-bay-lake-huron-_5a4c358eb7d7bcb74fe9482b_pdf>

(6) Environment and Climate Change Canada, Manitoba Agriculture and Resource Development. (2020). State of Lake Winnipeg (2nd edition). <https://www.gov.mb.ca/water/pubs/water/lakes-beaches-rivers/state_lake_wpg_report_tech.pdf>

(7) The distribution, density, and biomass of the zebra mussel (Dreissena polymorpha) on natural substrates in Lake Winnipeg 2017-2019. Depew, D., Krutzelmann, E., Watchorn, K., Caskenette, A., Enders, E. *Journal of Great Lakes Research* **2021** <https://doi-org.uml.idm.oclc.org/10.1016/j.jglr.2020.12.005>

(8) Beasley, D. E., Koltz, A. M., Lambert, J. E., Fierer, N., & Dunn, R. R. (2015). The Evolution of Stomach Acidity and Its Relevance to the Human Microbiome. *PloS one*, 10(7), 1-12. [https://doi.org/10.1371/journal.pone.0134116](https://doi.org/10.1371/journal.pone.0134116%20)

(9) Thorp, & Covich, A. P. (2001). *Ecology and classification of North American freshwater invertebrates* (2nd ed.). Academic Press.

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# Experiments

##### Molecular Biology (DNA)

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##### BioBrick Assembly

##### PCR to generate 5' modification

If the vector is a plasmid there will generally be a region where the are multiple cloning sites. The insert does not typically have a cloning site built in unless they are left over from previous cloning. PRC can be used to generate a large amount and insert and add the appropriate cut sites.

[PCR Protocol](https://static.igem.wiki/teams/4323/wiki/procedures/pcr.pdf)

##### Resctriction Enzyme Digest

Separately incubate both pieces of DNA (vector and insert) with two restriction enzymes. The vector and insert must be designed such that they have matching sticky ends. It is common to deactivate the restriction enzymes by heat denaturation once sufficient time had passed.

[Restriction Digest](https://static.igem.wiki/teams/4323/wiki/procedures/digestion.pdf)

##### Ligation

Now that both pieces of DNA have matching overhangs it is time to combine them and form the desired recombinant product.

[Ligation](https://static.igem.wiki/teams/4323/wiki/procedures/ligation.pdf)

##### Transformation and Selection

Hopefully some of the recombinant products made their way into the cells. Antibiotic selction will filter out any cells that don't have a plasmid. Unfortunately, those plasmids could be empty...

[Transformation](https://static.igem.wiki/teams/4323/wiki/procedures/transformation.pdf)

##### DNA Extraction "Mini-Prep" and Gel Solubilization

The process of isolating DNA from a matrix. In our case it is either an *E.coli* cell lysate of an agarose gel.

[MiniPrep Buffers](https://static.igem.wiki/teams/4323/wiki/procedures/mini-prep-buffers.pdf)

[Mini Prep Protocol from Fisher Scientific](https://static.igem.wiki/teams/4323/wiki/procedures/mini-prep-kit.pdf)

[Gel Extraction from Invitrogen](https://static.igem.wiki/teams/4323/wiki/procedures/agarose-gel.pdf)

##### Agasore Gel Electrophoresis

Agarose Gel Electrophoresis separates out the DNA molecules by size. This protocol was placed at the end of the workflow, but in reality, it can move around a little. This is a very versatile technique, we used it for the following.

After DNA extraction you perform a small digest and run the result on a gel. This allows you to identify if the colony you extracted the DNA from actually has the desired recombinant product.

After PRC it is common to run the result on an agarose gel. Since the size of the PCR product is known you can see if the PCR reaction was successful. Furthermore, this serves as a method to purify the DNA as they will be separated out by size. Cut out the desired and extract the DNA from the gel.

[Agarose Gel Electrophoresis](https://static.igem.wiki/teams/4323/wiki/procedures/agarose-gel.pdf)

##### Gibson Assembly

##### PCR to Linearize Vector and Generate 5' Overhang

Gibson assembly relies on sequence complementarity between the peices of DNA being joined. Unless the DNA has been specifically preformatted you will need to add the complementarity to the 5' end by PCR.

[PCR - Plymerase Chain Reaction](https://static.igem.wiki/teams/4323/wiki/procedures/pcr.pdf)

##### "One Pot" Gibson Assembly Reaction

The Gibson Reaction is an isothermal method: mix the following reagents, DNA with compatible overhangs, 5'-> 3' exonuclease, DNA polymerae and DNA ligase,

[Gibson Assembly Reaction](https://static.igem.wiki/teams/4323/wiki/procedures/gibson.pdf)

##### Molecular Biology (Proteins)

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##### General Protein Expression

For studying the proteins alone one of the simplest options is the ITPG induced T7 polymerase system.

[Protein Expression using the T7 system](https://static.igem.wiki/teams/4323/wiki/procedures/express-protein.pdf)

##### SDS-PAGE

[Preparing SDS-PAGE Gels](https://static.igem.wiki/teams/4323/wiki/procedures/sds-page-gel-prep.pdf)

[SDS-PAGE Sample Prep and Running](https://static.igem.wiki/teams/4323/wiki/procedures/sds-page.pdf)

[SDS-PAGE for UNILaussane](https://static.igem.wiki/teams/4323/wiki/technical-docs/sds-unil.pdf)

##### Affinity Chromatography (His-tag)

The most common method for protein purification is chromatography. By the use of a variety of stationary phases, proteins can be separated based on their size, hydrophobicity, isoelectric point or based on specific interaction. In our case, we used immobilized metal affinity chromatography (IMAC). Procedures below are for eGFP, but it is more or less the same with all His-tagged proteins.

[General example of IMAC](https://static.igem.wiki/teams/4323/wiki/procedures/production-and-purification-protocol.pdf)

[IMAC on AKTA start](https://static.igem.wiki/teams/4323/wiki/procedures/akta-procedure.pdf)

##### Verification of Function

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##### Fluorescence Assays

The file below describes the general procedure for both the phospate sensor and the nitrate sensro. The assay perfomed for the lehtbridge high shcool are very simmilar.

[Fluorecent Expression assay, genearl procedure](https://static.igem.wiki/teams/4323/wiki/procedures/96-fluo.pdf)

##### TAKEM4 buffer - Lethbridge High School

**10X TAKEM4 buffer**: 500 mM Tris pH 7.5,700 mM NH4Cl, 300 mM KCl, 10 mM EDTA, 40 mM MgCl2

##### Bioassays

The bioassays were all conducted by UNILausanne, the assay conditions are described below.

[Bioassy info found here](https://static.igem.wiki/teams/4323/wiki/unil-partner-ship/collaboration-with-manitoba.pdf)

##### Complete Workflows and GenBank Files

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##### Complete Workflows

[Nitrate Sensor](https://static.igem.wiki/teams/4323/wiki/technical-docs/no3-complete-bba-k216005.pdf)

[Phosphate Sensor](https://static.igem.wiki/teams/4323/wiki/technical-docs/po4-complete-bba-k1682012.pdf)

[Oxygen Sensor](https://static.igem.wiki/teams/4323/wiki/technical-docs/o2-bba-k239005.pdf)

##### Annotated DNA sequences as .gb (sensor and toxins)

[Plasmids](https://zenodo.org/record/7138051/files/zebrazap_2022_plasmids.zip?download=1)

[Primers](https://zenodo.org/record/7138051/files/zebrazap_2022_primers.zip?download=1)

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# Results

The main results of the ZebraZap

##### Truncated FitD "mini FitD"

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The truncated FitD functions as intended, albeit with slightly lower expression levels than desired. Detection by SDS-PAGE so has so far not been possible, despite many attempts, with any of the FitD produced by UNILausanne or UManitoba. The expression of these proteins clearly has room for optimization. Using an expression-plasmid could be a good first start, but we learnt this year that this is not always enough. Given the low concentrations apparently involved, liquid chromatography-tandem mass spectrometry is a promising technique. This could be done on the cell lysate and for homogenized mussel tissue to provide conclusive evidence and shed much light on the issue.

The modified FitD expressed in  *E.coli* has demonstrable a reproducible activity against Quagga Mussels, only a few graphs were shown for the sake of clarity. It could be noted that a total of 16 bioassays were conducted for FitD, as well as aerolysin disscussed later. The figure below shows all conditions tested. We chose only to show the tanks subject to the OD600 cell lysate.

For FitD expression, the longer the cells are allowed to grow as measured by OD*600* the greater the toxicity observed. Cells taken from cultures with low optical density have drastically reduced toxicity after OD600 = 0.5. Another trend is that the cell lysate is generally more toxic than the whole cells. Lastly, for FitD only the induced cells have any significant toxicity. We only chose to report a small fraction of the toxicity data to avoid making any premature conclusion. A true compairison would require usto screen for LD50, with know protein concentration. Note LD50 refers to the dose requried to kill half of exposed organisms.

![](https://static.igem.wiki/teams/4323/wiki/aerolysin/show-dont-tell.png)

Figure 1. Summary of the different parameters examined during the bioassays. The optical density refers to the optical density of the culture used when a small sample of that culture what introduced to the mussel tanks. In total there were 4 separate flasks and 16 tanks of mussels during each assay. Considering modified FitD and aerolsyin the is a total of 32 trials for the UManitoba constructs.

The truncation does not appear to have diminished the toxicity as shown by comparison to the full-length product produced by the native organism. As shown by the figure below. We do not consider the data below suggestive of improved toxicity, especially since the exact concentration of both products is not known. What effect the truncation may have had on the specificity of the toxin is not known and would need to be assessed.

![](https://static.igem.wiki/teams/4323/wiki/fitd/fitd-toxicity.png)

Figure 2. The induction of FitD is clearly the cause of the toxicity, the mussel are relatively healthy in the aquariums and the *E.coli* lysate itself is not toxic.Results obtained from tanks treated with cell lysate from cultures with high optical density OD600=1.

![](https://static.igem.wiki/teams/4323/wiki/fitd/fitd-toxicity-against-wild-type-final.png)

Figure 3. The Truncation does not appear to have hindered the toxicity.Results obtained from tanks treated with cell lysate from cultures with high optical density OD600=1.

##### Aerolysin as a Molluscicide

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The protein aerolysin has been shown to be a candidate molluscicide. *E.coli* transformed with the aerolysin construct but not induced, were found to be very toxic to quagga mussels. Our optimism is based on the fact that, they outperformed every other treatment examined by team UManitoba and UNILussane. The comparatively poor performance demonstrated by the induced samples is believed to be a consequence of the chassis being targeted by the protein it was forced to express. The specificity of the toxin remains to be seen. Specificity assays are likely more important than optimizing expression at this stage. Recommendation to test against non-invasive mussels. Current our intuition is that it may be more suitable as a general molluscicide with something else providing specificity.

Regarding the different expression parameters aerolysin protein showed very hight toxicity at all contions tested. The protein binds surprisingly tightly to the Nickel resin, but we were able to purify the protein and show expression.

![](https://static.igem.wiki/teams/4323/wiki/aerolysin/aerolysin-purification.png)

Figure 4. His-Trap Ni-column purified ACT. L: ladder, C5: 10 % B wash, D1: fraction of gradient at ~30 % B, D4: fraction of gradient at ~50 % B, D8: elution at 100 % B. A buffer: 20 mM Tris-HCl, 200 mM NaCl, pH 8.0; B buffer: 20 mM Tris-HCl, 200 mM NaCl, 300 mM imidazole, pH 8.0. Arrow: purified ACT protein, MW: 55.2 kDa.

![](https://static.igem.wiki/teams/4323/wiki/aerolysin/aerolysin-toxicity.png)

Figure 5. The greatest toxicity is observed for non-inudced cells, note that the Induced cell are stil quite toxic. Results obtained from tanks treated with cell lysate from cultures with high optical density OD600=1.

##### Sensors

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Achieving reliable sensing in the form of eGFP output proved to be more difficult than anticipated. Over the course of the project we built and attempted to measure the out put in response to NO3, PO4, O2 and pH. We were able to assay and reliably measure NO3 and PO4. The assays are explanined in detail in the experiments section.

![](https://static.igem.wiki/teams/4323/wiki/promoter-stuff/nitrate-hill-function.png)

Figure 6. Nitrate responsive promoter BBa\_ clonned into. Experimental experience and results added to the iGEM registry.

![](https://static.igem.wiki/teams/4323/wiki/promoter-stuff/phosphate-hill-function.png)

Figure 7. Nitrate responsive promoter BBa\_ clonned into. Experimental experience and results added to the iGEM registry.

##### Summary

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The sensors in this project were intended to add a level of redundency to soften any potential off-target effect the toxins may have. Nitrate and Phospate function as intended, but are not appropriate for application. The pH and oxygen sensors did not function as intended. At this point we would recommend that future experimenters device an alternate approach.

Both the modified FitD and the aerolysin appear to be very promising. A short list of next steps would be to optimized expression, critically evaluate toxicity LD50 , perform more detailed analytics and think about moving to procaryotic algae. The experiments conducted have a high chance of being expanded into a continued project, what form this will take has yet to be determined.

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