iGEM 2023 — Team barcelona-ub

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![](https://static.igem.wiki/teams/4770/wiki/titols/music-soundcloud-banner.png)

In July, we started our lab journey at the Centre for Research in Agricultural Genomics (CRAG).
Our first challenge was gene design. We had to optimize our genes to be more compatible with the codon guide of Chlamydomonas reinhardtii, but also to have a low enough content of GC nucleotides so they could be synthesized.
To solve this, we had to design a codon optimizer for our genes that took into account both GC content, and the natural codon proportions of Chlamydomonas reinhardtii.
All of these improved genes can be found in our Mo-Clo part collection, on the Parts page of our wiki.
Eventually, we were able to obtain a compatible sequence, order our genes, and start performing trial transformations.
Simultaneously, we were testing non-transformed Chlamydomonas compatibility with nitrate concentrations and wastewater components, to better understand their survival and growth under these conditions; and also to have a control with which to compare our transformed algae’s performance.
Last of all, we learned about cytokinin effects, detection, and quantification, which are very intricate time-consuming methods.

# Protocols

# Lab Notebook

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![](https://static.igem.wiki/teams/4770/wiki/titols/engineering-success.png)

Synthetic biology can be described as the genetic redesign of living organisms to fulfil a purpose, and with synthetic biology comes bioengineering.

Throughout the whole process of creating AlgaGenix we have used the bioengineering principles while designing the different parts of our genetic circuits and therefore understand, modify and control the living systems we are working with. The engineering design cycle (Define, Ask, Design, Build, Test, and Learn) serves as the basis for every bioengineering process and our project is no exception.

One significant advantage of using this cycle as an outline for our project development is that once you start your first engineering cycle, if anything unexpected happened you can re-enter it at any point to further develop and improve the process and your final product.

![](https://static.igem.wiki/teams/4770/wiki/engineering/captura-de-pantalla-2023-10-12-133917.png)

# Define

Identifying and defining the problem is a crucial first step for every project.

Our project was primarily motivated by one of our main local problems: water nitrification.

Due to climate crisis and intensive human activity such as farming, agriculture, industry and sewerage, good-quality water has never been so scarce. Our water reservoirs are at all-time low levels; however, the greater issue is that this little water that we still have remaining is of really poor quality, its main pollutants are nitrates, which are building up to create a significant strain on our resources.

![](https://static.igem.wiki/teams/4770/wiki/engineering/presentaci-fellowtalks-5.png)

This issue has a significant effect on society, studies show that more than 40% of Catalonia’s municipalities- around 3 million people\*- are affected by this problematic as levels of nitrates in local aquifers skyrocket. Finding a solution is urgent as nitrates have a major negative impact on health; oxidizing the iron molecules from the hemoglobin in our blood causing a deficiency in the oxygen transport through our bodies.

![](https://static.igem.wiki/teams/4770/wiki/engineering/implementation.png)

# Ask

Now that we have the problem we aim to tackle clearly defined, the next step is to ask ourselves "How can we help? How can we make a difference? What are the most urgent points that need to be addressed?"

Our goal is to design a functional, affordable unit able to absorb and convert pollutants, so that any person affected by contaminated waters of this nature can feel safe while drinking.

The solution we came up with is AlgaGenix, where we aim to improve the natural ability of the microalgae model organism Chlamydomonas reinhardtii to absorb nitrates so that these are removed from polluted water, and we hope to go one step further and use these absorbed nitrates to produce a value-added product: cytokinin-rich biomass, which can later be used as a fertiliser, while improving water quality.

To see an extended explanation of the questions our project revolves around, please visit Our approach.

# Design

Finding the key genes involved in the nitrates absorption route and cytokine generation was the first step in the construction of AlgaGenix.

Five genes can be distilled from the literature: Nitrate Reductase, Nitrites Reductase and Glutamate Synthetase for nitrates and IPT and LOG for cytokines. From now on, the different vectors containing these genes will be referred as “pathway genes”.

![](https://static.igem.wiki/teams/4770/wiki/engineering/engineering/implementation-2.png)

Please visit the parts page for further details on how each gene functions and is used in our project.

Before getting started with the enhancement of nitrates assimilation and cytokine production, we wanted to ensure that we would be able to detect said changes in the concentration of nitrates in polluted water.

To this end, we decided to synthesize a Nitrates Sensor similar to that of Crozet et al. (2018), yet with a small change: our sensor doesn't have an antibiotic resistance gene linked to the reporter (GFP in the case of the paper), instead, we decided to make distinct cassettes, one containing mVenus (pCM1: mVenus), one with mCherry (pCM1: mCherry), as well as an individual cassette for hygromycin resistance (pCM1: HygroR).

pCM1 will include a promoter (constitutive or nitrates-dependant), the gene and a constitutive terminator.

![](https://static.igem.wiki/teams/4770/wiki/engineering/presentacio-n3-pptx-4.png)

Resistance cassette will be used for colony selection and ensuring the assembly of the vectors. Moving into the fluorescent cassettes, the purpose of having two reporters is to be able to measure the plasmid's expression level depending on both, the location of its insertion within the Chlamydomonas genome using a constitutive promoter (PPSAD assembled into pCM1: mCherry) and at the same time measure the expression dependent on nitrates using a promoter highly induced on nitrate (PNIT, paired in pCM1: mVenus).

To summarize, on one hand, the mCherry signal is expected to be showing constitutively and its intensity will depend on the part of the genome it is inserted in. On the other hand, we will see mVenus’s signal in the presence of nitrates, thanks to the PNIT promoter.

Later, these 3 cassettes will be combined to create the final Level M of the sensor and included in the Level Ms of the different pathway genes.

![](https://static.igem.wiki/teams/4770/wiki/engineering/presentacio-n3-pptx-5.png)

After designing our sensor, we proceeded to design the pathway genes.

We decided to use individual genes, paired genes, and a combination of all genes. All of these Level M would be created with the same Level 1 included; this would eliminate the need for several Level 1s with the same gene to be produced and allow for the most effective use of our resources.

Level 1s are transcriptional units with different pieces available for assembly, which gives us a wide range of options to connect the different parts of our vector.

Usually, Level 1s have an A1-B2 region that includes promoter+5’UTR, B3-B5 with coding sequences, and B6-C1 containing terminator+ 3’UTR.

All AlgaGenix’s Level 1s will be put together using the following strategy:
- A1-B2: PPSAD promoter including 5’UTR region
- B3: coding sequence for the target gene which may be: NR, NiR, GS, IPT or LOG.
- B4: this piece will be an F2A self-cleaving peptidase that will act as a link with the next B5 piece.
- B5: will include a NanoLuc reporter
- B6-C1: TPSAD terminator including 3’UTR

There is one exception that slightly differs from this scheme, with the Cp-GS Level 1 we have reduced the space occupied by the PPSAD promoter and 5’UTR to be able to fit a chloroplast transit peptide as a B2 piece without having to redesign all the other parts.

The goal of these Level 1s is to overexpress the genes.

![](https://static.igem.wiki/teams/4770/wiki/engineering/presentacio-n3-pptx.png)

Design of level M:

All Level M also follow the same strategy only changing the pathway genes that are enhanced by changing the gene of Level 1 that we would be assembling into each Level M.

General chassis is formed by four cassettes:
- Resistance cassette: pCM1 HygroR
- Gene cassette: pCM1 NR/NiR/GS/Cp-GS/IPT/LOG
- mVenus cassette: pCM1mVenus (nitrate-dependant)
- mCherry cassette: pCM1mCherry (constitutive)

We have organized the Level Ms into three big groups depending on the combination degree of the pathway genes:
- Individual genes
- Pair genes
- Combination of all genes

Position of the cassettes inside the Level M will vary depending on the combination degree:

##### Individual genes

The aim of the Level M is to confirm the expression and position of the integrated vectors.

It will have the resistance cassette (pCM1: HygroR) on position 4, the gene cassette (which can be pCM1: NR, NiR, GS, Cp-GS, IPT or LOG) on position 5, followed by the two reporter cassettes; first mCherry (pCM1: mCherry with the PPSAD promoter) on position 6 and mVenus (pCM1: mVenus with the PNIT promoter) on position 7.

![](https://static.igem.wiki/teams/4770/wiki/engineering/presentacio-n3-pptx-1.png)

##### Pair genes

The goal is to see the effects of overexpressing the gene pairs of each step; NR+NiR being first steps of nitrates assimilation, and GS+CpGS final step of nitrates assimilation. Then, IPT+LOG for cytokine synthesis.

In this assembly it was important to think how to move the different pieces in order to make the less changes possible; the more pieces we can keep having in the same position as in the Level M for individual genes, the better. At the end, we have moved the resistance cassette to position 3 (this means we have to assemble it with the proper overhangs for position 3), NR, GS and IPT to position 4 but we are keeping, NiR, CpGS, LOG, mCherry and mVenus cassettes in the same positions

![](https://static.igem.wiki/teams/4770/wiki/engineering/presentacio-n3-pptx-2.png)

##### Combination of all genes

Lastly, to see the effect of overexpressing all genes at the same time, the final Level M would be the combination of all pair genes, the problem is that MoClo’s Level M doesn’t have enough positions to fit the resistance, the reporters and the cassettes of all genes in the same vector. It has up to 7 positions and we would need to fit 9.

To solve this, we decided that we would create 2 more resistance cassettes with resistance for different antibiotics and perform three different transformations:
1. First transformation with the NR-NiR paired assembly with HygroR
2. Second transformation with paired GS-Cp-GS with a different resistance.
3. Third and last transformation with paired IPT-LOG with another resistance.

![](https://static.igem.wiki/teams/4770/wiki/engineering/presentacio-n3-pptx-3.png)

Once we had all the cloning plans, we went ahead with adapting the sequences for MoClo.

We added to each MoClo piece its own unique overhangs in order to be assembled on the right backbone vector and in the right place. In addition, to make our sequences suitable for MoClo, we had to eliminate the recognition sites for BbsI and BsaI by changing them with synonymous codons.

The following step was to purchase the designed sequences. However, this is the point we first had to stop our engineering cycle.

*Chlamydomonas reinhardtii*’s genes have a characteristically high GC content, which made it impossible to synthesize them commercially.

# Building

In order to solve this huge obstacle, we designed an optimizing code.

*Chlamydomona reinhardtii* is a unicellular green alga that serves as a model organism for research in several fields. One challenge that researchers encounter when working with Chlamydomonas is its high GC content in its genetic code, which often leads to issues during gene synthesis due to repetitive sequences. Here, we present a Python script developed to optimize the sequences of Chlamydomonas genes with a very high GC content, making them more suitable for gene synthesis.

The initial step of the optimization process involves providing an input sequence with a high local GC concentration, which is characterized by repetitive sequences. This sequence served as the basis for further analysis and optimization.

To address the high GC content issue, we have designed a codon table that associates each triplet codon with its corresponding amino acid and a fractional value. This fractional value represents the optimized probability of using a particular synonymous codon. By utilizing this codon table, we can make informed choices for synonymous codons to reduce the overall GC content in the gene sequence.

One of the critical challenges when dealing with high GC content in Chlamydomonas gene sequences is the presence of repeated sequences. Repetitive sequences exacerbate the problem during gene synthesis, as they lead to an overabundance of guanine (G) and cytosine (C) nucleotides. Therefore, the script is designed to identify these repetitive patterns within the gene sequence.

The heart of the optimization process involves an iterative loop. The script repeatedly makes codon changes based on the codon table and monitors the resulting sequence for two criteria:

Acceptable GC content: The GC content of the sequence should be lowered to a level where gene synthesis becomes feasible. In the case mentioned, the target was to achieve a GC content that was below the problematic 66.30% threshold.

Reduced Repetition: The script aims to reduce the frequency of repetitive sequences to ensure that the synthesized gene is unique and non-repetitive.

The iteration continues until the sequence meets both criteria. In the case that the GC content and repetition issues persist, the codon changes are applied up to 10,000 times to arrive at an optimized sequence.

![](https://static.igem.wiki/teams/4770/wiki/engineering/communication-graphics-5.jpg)

In conclusion, the Python script described provides an effective solution to the challenge of high GC content in Chlamydomona reinhardtii’s gene sequences. By utilizing an optimized codon table and iterative optimization, the script can produce gene sequences with reduced GC content and minimal repetition, making them suitable for gene synthesis.

This approach ensures that Chlamydomonas genes can be synthesized without encountering the issues associated with high GC content and repetitive sequences, thus facilitating research and genetic engineering involving important model organisms.

# Test

To verify the sequences we extracted from the code, we confirmed that the GC content had been approved by the company that was responsible for synthesizing them. We then matched the sequences with the originals to see if the amino acids had not changed.

![](https://static.igem.wiki/teams/4770/wiki/engineering/grafiquet.png)

The company accepted the sequences for synthesis and the alignment was a success.

# Building

Once the sequences arrived, we continued with our second building step: the assembly of the constructs.

MoClo is based on the Golden Gate assembly and uses type II S restriction enzymes, BbsI is used to assemble pCM0s, BsaI is used to assemble pCM1s, and BbsI once more is used to assemble pCMMs.

All transformations were first done in E. coli to accelerate the process, because Chlamydomonas reinhardtii needs up to 10 days to grow, whereas with E.coli we could see colonies the following day after plating.

We successfully assembled and transformed both Level 1 and Level M of the sensor in E.coli and Chlamydomonas reinhardtii.

Moreover, we were able to assemble Level 1s of all the pathway genes, and currently working on transforming them into E.coli and Chlamydomonas reinhardtii.

![](https://static.igem.wiki/teams/4770/wiki/engineering/level-1.png)

# Test

We tested the successful transformation of the sensor in Chlamydomonas reinhardtii:

![](https://static.igem.wiki/teams/4770/wiki/engineering/whatsapp-image-2023-10-12-at-04-51-45.jpeg)

![](https://static.igem.wiki/teams/4770/wiki/engineering/whatsapp-image-2023-10-12-at-04-51-57.jpeg)

We are currently checking its function and the transformation of pathway genes into Chlamydomonas reinhardtii.

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![](https://static.igem.wiki/teams/4770/wiki/titols/results.png)

Despite many failed transformations, and algae killed due to growth conditions, we were eventually able to obtain some data and make advancements on our project.

# 1. Parts collection, Engineering Success and Software

You can find a detailed explanation about the Parts Collection and the Engineering success in the Parts and Engineering sections of our Wiki.
In summary, our part collection includes two basic sets of genes:
- Set 1: Nitrate assimilation: Nitrate Reductase (NR), Nitrites Reductase (NiR), Glutamate Synthetase (GS), Cp-GS
- Set 2: Cytokinin production: IPT, LOG
The gens went through Level 1 and Level M assemblies.
In addition, we successfully transformed C. reinhardtii (and we demonstrated it) with a nitrate sensor, a new part that indicates the changes in nitrate concentration of the medium.

Finally, the Software description can be found in the Engineering section and its contribution is explained into the Contribution page.

# 2. Nitrate Assimilation in ideal conditions

This experiment’s main objective was discovering Chlamydomonas reinhardtii’s LM8523 and UVM4.

The data from this analysis comes from two different experiments.

##### 1. Nitrate assimilation (start date: 12/7/2023)

This was the first nitrate assimilation experiment we performed in chlamydomonas, and the goal of the experiment was to observe difference in growth rates, or potential inhibitions, from different concentrations of nitrate in the medium (Check protocol Determining the efficiency of nitrate uptake from the culture medium in two Chlamydomonas Reinhardtii strains).

For the first couple of days, no obvious differences were observed between the algae growth in different mediums, so we had to let the cultures incubate for longer.

![](https://static.igem.wiki/teams/4770/wiki/results/1-wiki-results-image-1.jpg)

This picture depicts the culture preparations immediately after preparation. On the left, UVM4 strains, and on the right, LM8523 strains. As it clearly depicts, there is no algal growth.

![](https://static.igem.wiki/teams/4770/wiki/results/2-wiki-results-image-2.jpg)

This picture depicts the culture preparations 48 hours after preparation. On the left, UVM4 strains, and on the right, LM8523 strains. As it clearly depicts, wells with 105, 90 and 1mM barely present any growth. However, wells 15, 45, 280 and 385mM present algal growth, specially the UVM4 strain. The white wells remain uncontaminated.

The results in this experiment are not entirely reliable, as we obtained some very unexpected results:
ߦThe wells with highest nitrate concentration mediums (280mM and 385mM) had the highest growth rate of all cultures, higher even than optimal concentration nitrate mediums (1 - 15 mM).
- These results were very strange to us, as we expected those concentrations to be too high, and we expected them to inhibit algal growth instead.
- We theorised that what might have happened is that the nitrate medium preparation was compromised due to the high concentration of the nitrate solution we used to prepare the mediums, which caused the creation of KNO3 crystals in the solutions. We believe this might have changed the concentration of the prepared solutions.
ߦ There was a significant lack of growth in nitrate concentration mediums of 90mM and 105mM. We could not quite explain why this happened, or how to justify the previous results. Which is why we repeated the experiment with some improvements a few days after.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-3.png)

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-4.png)

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-5.png)

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-6.png)

The graphs above show the measurements we took after 48h of the culture preparation, for both fluorescence and OD absorbance, as well as UVM4 (upper line) and LM8523 (lower line) strains.

The most significant data we can extract from these graphs is the significant decrease of growth, that in the fluorescence samples is for growth mediums 90-105mM; but OD absorbance gives us different values.

This inconsistency in data, and the lack of literary support for these results, is another reason why we need to repeat the experiment.

One stable trait about the graphs, however, is that UVM4 seems to grow faster than LM8523, as both values for fluorescence and OD absorbance are higher in the UVM4 strain.

The results of this experiment were inconclusive, so we had to repeat it, so we could observe whether this pattern was recurrent.

##### 2. Nitrate assimilation (start date: 19/7/2023)

The procedure for this experiment is found in the protocol Determining the efficiency of nitrate uptake from the culture medium in two Chlamydomonas Reinhardtii strains.
This time, to better track the algal growth in the nitrate enriched culture mediums, we decided to take more measurements: At 2h, 50h, 120h and 168h.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-7.png)

96 well plate we used for the 2h measurements. At this stage, we still can’t observe significant algal growth.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-8.jpg)

The algae cultures after the span of 24h. Still no observable growth. On the left are LM8523 strain cultures, and on the right, UVM4 strain cultures.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-9.jpg)

The algae cultures after the span of 48h. On the left are UVM4 strain cultures, and on the right, LM8523 strain cultures. We observe growth within the expected results, except for W2 of 15mM LM8523, where we forgot to add algae sample, so we won’t be accounting it when we make calculi, as it is going to behave more similarly to a white sample and could affect the accuracy of our data. This change is implemented for all the measurements we did.
This same day, we performed another analysis for their growth with the SpectaMax (50h measurement). You can find the instructions in the protocol “Measuring Chlamydomonas reinhardtii growth by using the SpectaMax”.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-10.jpg)

Update on the algae cultures at time 120h. On the left, UVM4 cultures, and on the right LM8523 cultures. There is no significant observable change, as none of the mediums that didn’t present activity at time 50h presents it now either. However, the wells that do present algal activity, seem to have darkened, meaning the algae have continued growing.
This same day, we performed another analysis for their growth with the SpectaMax (120h measurement). You can find the instructions in the protocol “Measuring Chlamydomonas reinhardtii growth by using the SpectaMax”.
During the preparation of the 96-well plate we observed some irregularities in the wells:
- UVM4 105mM cultures presented formations of clumps of algae in the medium, some had precipitated.
- UVM4 280mM cultures became less clear, and adopted a more translucent appearance.
- Some of the wells appeared to have less volume, a sign of the well mediums potentially drying out. This is why from now onward we will be sealing all the plates with plastic wrap, to avoid losing moisture from the sample.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-11.png)

Image of the 96-well plate preparation we used for the 120h measurement in the SpectaMax. There are two replicates for each of the wells, as well as standard samples (ST, 90mM nitrate concentration without algae).

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-12.jpg)

Update on the algae cultures at time 144h. On the left, UVM4 cultures, and on the right LM8523 cultures. The plastic wrap hinders the observation of the writing, but the well plates are distributed exactly as the progress picture we took of the cultures at 120h.
There is no significant observable change, and it seems like the algae culture has stabilised (reached maturity).

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-13.jpg)

Update on the algae cultures at time 168h. On the left, UVM4 cultures, and on the right LM8523 cultures. The plastic wrap hinders the observation of the writing, but the well plates are distributed exactly as the progress picture we took of the cultures at 120h.
This is the last update picture as time 168h is the last measurement we took of the experiment. Again, there are no significant observable changes between these cultures and the 144h cultures.

We prepared the last 96-well plate to measure OD absorbance and fluorescence.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-14.png)

Image of the 96 well plate we prepared for the 168h measurements at the SpectaMax.

For data in all the values obtained from measures with the SpectaMax, please refer to our lab notebook.

##### Conclusions

We extracted all the data from the previous experiments and prepared four graphs, each for OD absorbance/fluorescence and LM8523 / UVM4.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-15.png)

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-16.png)

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-17.png)

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-18.png)

From this initial data we can determine that for samples containing 280 and 385mM nitrate concentration in their medium, there was barely any growth, to the point of resembling white sample levels, in both UVM4 and LM8523 strains.
For nitrate concentrations; 1mM, 15mM, 45mM and control (0mM), seem to perform the best and present out of all the cultures. In some of the cases, even 105mM presents good performance.

After completing growth analysis of the Chlamydomonas culture mediums, we used the remaining of the mediums to follow the protocol titled “Measuring nitrate levels from a liquid medium”.
The first step was the preparation of the nitrate calibration curve, from which we obtained the following graph, from were we extracted the formula of the tendency line so we could calculate the concentrations of the algae growth mediums:

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-19.png)

However, upon using the value of the calibration curve to obtain the final nitrate concentrations of our algae we soon realised that there must have been a mistake during the procedure, as the results are the ones from the table below:

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-20.jpg)

The data is clearly inconsistent, especially because of the presence of negative value concentrations. After revising our data and calculi, we have found an error in the calculi for the preparation of the sample dilutions. It seems as though we didn’t dilute the highest concentrated growth mediums enough, and also like we didn’t get enough data points to calculate the nitrate calibration with enough accuracy.
We are improving the protocol, re-calculating and re-writing it, so that when we eventually repeat this experiment we get usable data.
Therefore, we haven’t been able to accurately get data on nitrate absorption performance of Chlamydomonas reinhardtii strains LM8523 and UVM4, but we have been able to determine the ideal nitrate concentration range for their growth; [1, 45] mM.

# 3. Nitrate Assimilations in artificial wastewaters

The intent of this experiment, in the beginning, was to determine the nitrate uptake from the culture medium when samples of Chlamydomonas reinhardtii were cultivated in a medium that recreates wastewater (artificial wastewater).
The protocol used for the preparation of this experiment is listed under the title “Determining the efficiency of nitrate uptake from the culture medium in two Chlamydomonas reinhardtii strains”.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-20.jpg)

Initial picture of the culture mediums immediately after preparation of the plates (time = 0h). On the left, UVM4 strains, and on the right, LM8523 strains. At this time, there are no signs of algal activity.
Over the course of the days, we noted no significant difference.
On the final day of the experiment, we took one last progress photo that demonstrates no evolution in the algae cultures, as the cultures at t=120h are the same as in t=0h.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-21.jpg)

Image of the wastewater nitrate assimilation experiments at t=120h. We observe no difference from the beginning of the experiment, and all the culture mediums behave the same as the white wells.

##### Conclusions

Given the results in this experiment (lack of any sign of algal growth), we decided not to measure the nitrates in the medium, as they would yield us no significant data. Our objective for the experiment was determining whether Chlamydomonas reinhardtii strains UVM4 and LM8523 ability to absorb nitrates from the medium was, to some degree, hindered by the wastewater conditions.
Instead, we were able to determine that at least one of the components from artificial waste water inhibited the development of algae. We were also able to rule out that it was caused by the nitrates, as the simultaneous experiment we were conducting (Nitrate assimilation in ideal conditions) had the same nitrate concentrations and did present growth.

From the results of this experiment, we decided it was necessary to determine which of the artificial wastewater components was inhibiting the algae proliferation, and instead of measuring the nitrate concentration of the medium, we proceeded to design a follow-up experiment: Wastewater inhibiting component.

# 4. Wastewater inhibiting component

After demonstrating that algae did not grow in artificial waste water conditions in our previous experiment (Titled Nitrate assimilation in artificial wastewater, that can be followed with protocol ‘Determining the efficiency of nitrate uptake from artificial wastewater culture medium in two Chlamydomonas reinhardtii strains), we started a new experiment to determine which of the wastewater components presents inhibition towards Chlamydomonas reinhardtii’s growth.
The protocol for this experiment is titled ‘Determining the effect of wastewater components on two Chlamydomonas Reinhardtii strains' growth’.
This protocol is modified to test mediums with one sole component from the artificial wastewaters. It is also designed to really control algal growth, as this is what we are most interested in discovering with this experiment, finding the inhibiting component of algae proliferation.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-22.jpg)

96-well plate prepared at t=0 to measure initial activity of algae in the mediums. No activity can be observed. UVM4 strain is in the top rows, whilst LM8523 is in the bottom rows.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-23.jpg)

Wastewater component culture update at t=24 hours. We still cannot observe indicators of algae growth. On the left, LM8523 cultures, and on the right, UVM4 cultures.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-24.jpg)

48 hour update on the cultures. UVM4 strain on the left and LM8523 strain on the right. There are clear signs of algae growth in all wells, except for the white wells and compound J wells (Na2EDTA·2H2O). It seems we have already found the growth inhibitor, but we will still study the development of the experiment.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-25.jpg)

We took a second measurement at t=48h, and this is the 98-well plate structure that we used in the SpectaMax. UVM4 is in the upper rows and LM8523 in the lower rows.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-26.jpg)

Update picture on the cultures at time 120h. On the left, UVM4 cultures, and on the right, LM8523 cultures. We observe further growth, but some of the wells have lost moisture and have started drying out, the most clear examples is F compound of LM8523, J compound of UVM4 and B compound of UVM4.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-27.jpg)

This is the 96-well plate we used for the 120h measure of algal growth. For the wells that were starting to dry, we decided to use half the volume of the sample and then multiply the data of those wells times two during the analysis. To prevent further drying, we will seal the well plates with plastic wrap moving forward.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-28.jpg)

Update picture on the cultures at time 144h. On the left, UVM4 cultures, and on the right, LM8523 cultures, same disposition as the update picture for 120h. We observe stable growth, and it also seems as though compound H well of UVM4 has started drying out.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-29.jpg)

Update picture on the cultures at time 168h. On the left, UVM4 cultures, and on the right, LM8523 cultures, same disposition as the update picture for 120h. We observe stable growth, in all wells except for the white wells and the J-compound wells.
At this stage, it is safe to assume that compound J (Na2EDTA·2H2O) is what was inhibiting the algae growth.

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-30.jpg)

98-well plate preparation for the algal growth preparation at t=168h. This is the last measurement of the experiment. UVM4 cultures on top, and LM8523 cultures on the bottom. Again, the only wells where we don’t observe any algae growth are the whites and J-compounds.

##### Conclusions

From the very first stages of algal growth we determined that Na2EDTA·2H2O was most likely the inhibitor of the algal growth, and the fluorescence evolution values demonstrate it clearly, to the degree that the sample that contained Na2EDTA·2H2O in its medium has values that resemble those of the white sample.
We were therefore successful in detecting the main lone-acting inhibitor of Chlamydomonas reinhardtii growth from the components of artificial wastewaters.
We observed some unexpected behaviours in certain mediums that were able to surpass the growth rate of the control sample, especially at the peak of the algal exponential growth (120h).

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-31.png)

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-32.png)

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-33.png)

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-34.png)

ߦFor UVM4, the components were: CaCO3, NaCl, KH2PO4, MnCl2·4H2O, CoCl2·6H2O, FeCl3·6H2O and CaCl2·2H2O. All the components are ordered from highest growth boost to lowest growth boost above the control sample.
ߦFor LM8523, the components were: CaCO3, MnCl2, NaCl, CoCl2·6H2O, KH2PO4, FeCl3·6H2O, MgSO4·7H2O, ZnCl2. All the components are ordered from highest growth boost to lowest growth boost above the control sample.
- It is important to note that whilst components CaCO3, MnCl2, NaCl, CoCl2·6H2O and KH2PO4, reached algae growth peak at 120h, they proceeded to diminish in value at the next measurement (168h)
- In contrast, components FeCl3·6H2O, MgSO4·7H2O, ZnCl2 had not yet reached the growth peak even at 168h.
We found the results of this experiment very intriguing, as the prospect of creating an enriching growth boosting culture medium for algae with fewer or cheaper components is very promising. We have therefore decided to study this phenomenon further by designing a new set of experiments, Culture medium enricheners, which can be followed in the protocols titled “Determining the effect of salt supplements on two Chlamydomonas reinhardtii strains’ growth”.

# 5. Culture medium enricheners

##### 1. Experiment 1

With the observed results from the ‘Wastewater inhibiting component’ experiment, we designed a new test concerning those salts that showed a growth boost of our microalgae:
- CaCO3
- FeCl3
- MnCl2
- NaCl
- ZnCl2
We designed 7 sets of mediums:
- 5 excluding one of those components in each
- Medium with every enrichener salt
- Negative control (TAP medium).
The procedure is described in the following protocol ““Determining the effect of salt supplements on two Chlamydomonas reinhardtii strains’ growth (1)”.
We wanted to discover if the mixture of the growth-boost components was favourable for algae growth and to sse which one of the enricheners was more determinant (both in UVM4 and LM8523 strains).

##### 1.1. Results analysis

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-35.jpg)

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-36.jpg)

C → Control. Tap medium.
1 → TAP, CaCO3, FeCl3, MnCl2, ZnCl2
2 → TAP, CaCO3, FeCl3, NaCl, ZnCl2
3 → TAP, CaCO3, NaCl, MnCl2, ZnCl2
4 → TAP, NaCl, FeCl3, MnCl2, ZnCl2
5 → TAP, CaCO3, FeCl3, MnCl2, NaCl
6 → TAP, CaCO3, FeCl3, NaCl, MnCl2, ZnCl2

As observed in the graphs, mediums 2 and 3 showed a lower microalgae growth than the control for both strains.

When considering the LM8523 strain, medium 1 was the only one that showed a clear growth boost in comparison with the control. Mediums 4, 5, 6 present a similar growth pattern than the control.

For UVM4 strain, mediums 1 and 6 seem to have a better growth than the control, showing a surprisingly high boost at 192h.
Mediums 4 and 5 have a similar growth pattern as the control.

##### 1.2. Conclusion

As medium 2 and 3 didn’t contain either MnCl2 nor FeCl3, we need to conclude that these salts are indispensable for microalgae growth when mixed in a medium with other salts. Perhaps their presence contributed to damaging the cells in a determined way.

Medium 4, lacking CaCO3, showed us how this component might be also important in the salt synergy for microalgae growth.

The 2 strains don’t present an important difference in their reaction to the enricheners. In addition, it is likely that the observed boost of growth for mediums 1 and 6 was caused by contamination.

Nevertheless, the measures weren’t taken evenly and we should repeat the experiment in order to obtain valid results.

##### 2. Experiment 2

With the results from experiment 1, we have developed a new experiment selecting the best medium and salts combinations (mediums 1,5,6) for both strains, repeating their culture and creating 5 new medium combinations (all of them slight variations of mediums 1,5,6).

The procedure describing the medium preparations is included in the protocol “Determining the effect of salt supplements on two Chlamydomonas Reinhardtii strains' growth (2)”.

##### 2.1. Results analysis

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-37.png)

![](https://static.igem.wiki/teams/4770/wiki/results/wiki-image-38.png)

Mediums (if there is an added \*, it signifies an increase of the component concentration):
1. TAP (control)
2. TAP + MnCl2 + FeCl3 + CaCO3
3. TAP + MnCl2 + FeCl3+ CaCO3 + NaCl + ZnCl2
4. TAP + MnCl2 + FeCl3 + CaCO3 + NaCl
5. TAP + MnCl2 + FeCl3+ CaCO3 + ZnCl2
6. TAP + MnCl2\* + FeCl3\*+ CaCO3\*
7. TAP + MnCl2\* + FeCl3+ CaCO3
8. TAP + MnCl2 + FeCl3\*+ CaCO3
9. TAP + MnCl2 + FeCl3+ CaCO3

We observe how the controls present better growth for both strains. The peak is observed at 96h, and from that point, OD measures values decrease.
All mediums with increased concentrations show a bad growth performance, not even reaching half the growth of the control at the last measure.

Mediums 2, 5 and 5 in LM8523 strain don’t achieve their peak and growth decline. It would be possible that because of these supplements they decrease their growth speed but they still achieve the control maximum OD.
The UVM4 strain reaches growth peak at 168h, and decreases in OD during the several following measures.

##### 2.2. Conclusion

Results seem to show that Experiment 1 results are not reliable, as they do not present lasting continuity. We are therefore able to determine that the combination of these enricheners doesn’t seem to favour the growth of Chlamydomonas reinhardtii, for either of its strains.

Hence, it is better to culture this microalgae species in TAP medium rather than combine it with many other components. This addition will only decrease the growth speed of the culture.

# 6. Cytokinin effects on plants

##### Introduction

As a part of their university studies, two of our team members are taking a course on Biochemistry and Plant Physiology, where they had wet lab class related with the use of a biostimulant in Solanum lycopersicum, a tomato variety.
We decided that using the data of this experiment would be a great opportunity for us, as we would be able to gather information on the effect of cytokinins on the plant.
The experiment is based on the use of kinetin, a type of cytokinin. Literature reading let us determine they are associated with salinity-response stress mechanisms, which is of important interest to us, as those can often be a side-effect of drought.
Our goal for this experiment was using moderately salt water (result of mixing fresh water and sea water), and test whether it would be possible to irrigate crops in areas of high water stress with it.

The idea for this use of cytokinins and salt water stems from our team visit to Comunitat de Regants del Sindicat Agrícola de l’Ebre, who are an irrigation community really struggling with the effect of high salinity in their crops.

Therefore, we are using Mediterranean sea water as a reference for the concentration of the saline water, which has a concentration of 38 g/l.
We also discovered that 100mM of NaCl is considered “severe stress” for S. lycopersicum, whilst our saline water concentration reference is of 651mM.
To prevent conducting the experiment in a very harmful environment for the plan, we decided to dilute the sea water with fresh water, from which we can discover the adequate concentrations of cytokinins to use that can mitigate the effects of the high salinity water on the plant.

For example, in the Ebre region, sea water often infiltrates into the soil, thereby increasing its salinity. This is why we thought that implementing our project for a situation such as theirs, would be a beneficial situation for both of the members of the deal.

##### Action mechanism of Kinetin

The action mechanism of kinetin, like that of any other cytokinin, is not yet fully characterised. Despite this, a dual-component signalling model, similar to the bacterial system, has been established as the preferred model to explain the functioning of CKs (Shi et al., 2011).
In this system, CK acts by binding to histidine-kinase sensors that initiate a signalling cascade. This receptor, when autophosphorylated, transfers a phosphoryl group to a protein phosphotransferase (HPt), which at the same time transfers it to specific response regulators (RR) located in the nucleus.
There are two types of RR: those of type B, which are transcription factors that activate certain genes involved in plant development; and those of type A, which allow a negative feedback to be carried out to regulate this whole process.
Cytokinin response factors (CRFs) have also been found to act in parallel on certain downstream targets of cytokinins, either independently or in conjunction with B-type RRs.
Up to 11 CRFs have been characterised in Solanum lycopersicum, although its mechanism is not known to the same level as Arabidopsis thaliana. However, SlCRFs (Solanum lycopersicum CRFs) are expressed differently in a variety of plant tissues, and some are more sensitive to cytokinins than others.
In addition, abiotic factors such as salinity can also induce certain SlCRFs, suggesting that CRFs are directly involved in the response to salinity stress and that they may act more generally on other types of stress (Shi et al., 2011) (Ahanger et al., 2018).

![](https://static.igem.wiki/teams/4770/wiki/results/captura-de-pantalla-2023-12-04-a-las-21-10-55.png)

##### Experimental design

For this experiment, we have set two experimental groups, with 5 plants (n=5) in each one.
Each group will be treated with 5 mg of kinetin twice a week, in spray, and will be irrigated with a different salinity concentration: 108 mM or 217 mM of NaCl.
According to the consulted literature, 100 NaCl mM can be considered a severe salinity for plant development in Solanum lycopersicum, and 200 mM is practically lethal (Ahanger et al., 2020). From now on, we will be addressing our groups as “Severe” for the one with 108 NaCl mM, and “Extreme” for the one with 217 NaCl mM treatments.
The duration of the treatments was 3 weeks, when we also took the fruits and treated them to measure their biochemical parameters.

##### Results analysis

Firstly, we wanted to see the physiological effects of the sea-water treatment on our plants, so we measured the relative growth of the stem and the amount of chlorophyll in the leaves. The following graphs show how there are no significant differences between the «Severe» and the «Extreme» groups, which means that the amount of CK we applied was enough to neutralize the effects of high salinity.

![](https://static.igem.wiki/teams/4770/wiki/results/creixement.png)

![](https://static.igem.wiki/teams/4770/wiki/results/chlorophyll.png)

However, and still in the physiological department, the stomatal conductance did show significant differences between the two groups, which means that there were differences in water uptake.

![](https://static.igem.wiki/teams/4770/wiki/results/cond-est.png)

On the other hand, we also wanted to measure the quality of the fruits, so as to understand if plant survival was to the expense of yield and quality. In the fruits, we measured lycopene content, the amount of antioxidants and the sugar/acid ratio. The following graphs show that none of these parameters were significantly different between the two groups, so it is safe to say that the quality of the fruits was maintained.

![](https://static.igem.wiki/teams/4770/wiki/results/licop.png)

![](https://static.igem.wiki/teams/4770/wiki/results/rsa.png)

![](https://static.igem.wiki/teams/4770/wiki/results/ratio.png)

Lastly, we want to stress that the absence of a proper control is a clear issue that must be addressed when repeating this experiment. The main question one may ask is whether or not the differences in salinity really affected the development of the tomatoes, because there are no significant differences in practically any parameter. However, the stomatal conductance did show a significant difference between the two groups, which was expected as the plants with a higher salinity will have more difficulty to absorb water, so they’ll close their stomata.

Therefore, salinity did have an effect on the plants, but kinetin was able to neutralize its effect on the development of our tomatoes. However, a control for the kinetin treatment (no salinity stress but kinetin application), and a control of the salt concentration (tomatoes under salt stress but without kinetin treatment), are very much needed to fully ensure the preliminary conclusions of this experiment.

##### References

Van Trong L, Bao Thinh B, Hung N, Thi Huyen L, Thi Phuong H. Effects of Cytokinin on Physiological and Biochemical Indicators of Some Tomato Varieties (Solanum lycopersicum L.) Cultivated in Vietnam. Pakistan Journal of Biological Sciences : PJBS. 2020 Jan;23(11):1462-1472. DOI: 10.3923/pjbs.2020.1462.1472. PMID: 33274876.

Shi X, Gupta S, Rashotte AM. Solanum lycopersicum cytokinin response factor (SlCRF) genes: characterization of CRF domain-containing ERF genes in tomato. J Exp Bot. 2012 Jan;63(2):973-82. doi: 10.1093/jxb/err325. Epub 2011 Nov 7. PMID: 22068146; PMCID: PMC3254692.

Shani E, Ben-Gera H, Shleizer-Burko S, Burko Y, Weiss D, Ori N. Cytokinin regulates compound leaf development in tomato. Plant Cell. 2010 Oct;22(10):3206-17. doi: 10.1105/tpc.110.078253. Epub 2010 Oct 19. PMID: 20959562; PMCID: PMC2990126.

Kavitha, G., Kerketta, A., Topno, S. E., & Bahadur, V. (2023). Effect of Plant Growth Regulators on Cherry Tomato (Solanum lycopersicum var. cerasiforme). International Journal of Environment and Climate Change, 13(8), 581–586.

Ahanger MA, Alyemeni MN, Wijaya L, Alamri SA, Alam P, et al. (2018). Potential of exogenously sourced kinetin in protecting Solanum lycopersicum from NaCl-induced oxidative stress through up-regulation of the antioxidant system, ascorbate-glutathione cycle and glyoxalase system. PLOS ONE 13(9): e0202175.

Mohammad Abass Ahanger, Rayees Ahmad Mir, Mohammed Nasser Alyemeni, Parvaiz Ahmad, Combined effects of brassinosteroid and kinetin mitigates salinity stress in tomato through the modulation of antioxidant and osmolyte metabolism, Plant Physiology and Biochemistry, Volume 147, 2020, Pages 31-42, ISSN 0981-9428

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