iGEM 2023 — Team hust-ueve-upsaclay

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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![bg-notebook](https://static.igem.wiki/teams/4900/wiki/bg/bg-notebook.png)

## Our project

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#### 1.Recruit Team Members

From 2023/06/10 to 2023/07/01

We have recuit all of our members from HUST and we learn some basic expereiment skills with our teachers.

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#### 2.Development of Project Plan

From 2023/07/02

We have done a lot of human practice and articles reading to dicide the topic of our project and how to develop our project. We divided our group into 4 groups in order to better organize the teams

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#### 3.Wet Laboratory Activities

From 2023/08/15 to 2023/10/10

For group1, we mainly cultivate and prepare enough nematodes for other experiments.

For group2, we mainly prepare enough algae solution and measure the concerntration for other experiments to use

For group3, we mainly doing experiments to test that if there is chemotaxis between algae solution and cultivation solution

For group4, we mainly design the plasmid and test if the designed nemotodes have the ability to differentiate algae solution

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#### 4.Dry Laboratory Activities

From 2023/09/01 to 2023/10/08

We mainly designed a chip that can be used easily in real life environment

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#### 5.Building the Team Wiki

Fromm 2023/09/30 to 2023/10/13

We mainly build our Wiki to present all of our works about our projects.

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{% block title %}Engineering Success{% endblock %}
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![page-bg](https://static.igem.wiki/teams/4900/wiki/bg/bg-engineering.png)

#### Cycle one

##### Design

In order to detect and prevent water blooms at early stages, we hope that a single plasmid can be used to express β-galactosidase when *C. elegans* is under stress state, and make it to turn blue when exposed to certain concentration of microcystin so that the detection could be more efficient. We use existing components with independent functions to connect multiple components into systemic pathways with complete functions to construct complete plasmids to achieve our goal.

We set the stage for building the entire systemic pathway when we built the individual components. Firstly, we use Phsp-6 promoter to replace the original promoter Pbbs-1. It is the mitochondrial stress promoter, and may respond to increased reactive oxygen species (ROS) level caused by microcystin. Then we use modified LacZ to replace original TIR gene located on the downstream of Phsp-6. For the last step, we insert Met+SV40 NLS to guide LacZ gene into the nucleus with the way of homologous recombination.

##### Build

restriction endonuclease, ligase to construct the pathway, designed primers in strict accordance with the instructions, and carried out the operation process to reconstruct our plasmid. We used PCR technique to obtain gene segments from plasmids containing those sequences, and then verified and purified by gel recovery. Taking the replacement of Phsp-6 promotor as an example. We first used PCR to obtain the linearized sequence segment Phsp-6. The purified carrier and gene segment were verified by re-gel recovery. At last, the carrier and sequence segments were connected by T4 ligase, transformed and cultured. Finally, we validated the colony of bacteria to obtain our plasmid. The replacement of the TIR to modified LacZ was in the same previous way.

We attempted to ligate SV40 NLS directly to the carrier into complete plasmids using homologous recombinases at the last step. Firstly, we used PCR to obtain the linearized carrier Phsp-6: LacZ: SL2: mcherry and the segments. Then gel recovery was used to verify the purification of the carrier and fragments. Finally, homologous recombinase was used to directly link the linearized carrier.

##### Result

We try to use the reporter gene GFP, it can show whether the promoter hsp-6 works under stimulation of the MC-LR. In that case, we put the *C. elegans* under a fluorescence microscope to test the strength of the fluorescence and used software to calculate the intensity. At first, we designed 3 periods (each 24 hours) to check the fluorescence (Fig.1).

![fig-1](https://static.igem.wiki/teams/4900/wiki/engineering/engineering-fig1.png)

Fig.1 (a) It only shows the difference when the dealing time is 24h, in 48 and 72 hours there is no otherness. (b) We focus on the dealing time within 24 hours, but there shows no difference not only in 12h but also in 24 hours.

![fig-2](https://static.igem.wiki/teams/4900/wiki/engineering/engineering-fig2.png)

Fig.2. It shows that there are prominent differences between them.

##### Learn

Experimental operation has a great influence on the experiment. In the process of picking *C. elegans*, they cannot be put under stress. *C. elegans* under stress will emit fluorescence even if *C. elegans* do not be stimulated by microcystin, thus interfering with the experiment. However, we can use the promoter hsp-6 which can respond to increased ROS levels caused by MC-LR (microcystin), and it can play a role in other circumstances. As we have mentioned before, the current experiment does have some wobbles, but we believe that even after the iGEM competition, we will continue to promote our project, make efforts to use synthetic biology to improve the world environment and make rational use of resources.

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#### Water Collection

![experiment-water-collection](https://static.igem.wiki/teams/4900/wiki/experiment/experiment-water-collection.jpg)

**Configuration of algal culture medium:**

1. Take a 1L conical bottle and wash it
2. Add reagents in turn according to the recipe sheet
3. Sterilization with high pressure steam (121℃, 30min)
4. Pack into 500ml reagent bottle and store in refrigerator at 4℃

**Dilution of algal fluid:**

1. Remove the algal liquid from the 25℃ incubator and shake well
2. Take 1ml of the initial algal solution in a super clean table
3. The algal liquid was diluted about 100 times by gradient dilution method

**Algal concentration measurement:**

1. The algae concentration was measured using "count star"
2. The minimum measurement size of "count star" is 2 microns, while the diameter of microcystis aeruginosa is 3 to 7 microns
3. Shake the diluted algal liquid and transfer it to a counting plate (20 microliters per frame).
4. According to the measurement result picture, the most accurate result is retained to obtain the average value

#### *C.elegans*

![experiment-celegans-figure](https://static.igem.wiki/teams/4900/wiki/experiment/experiment-celegans-figure.jpg)

**Protocols for synchronization**

1. Day 1: Pick 10-15 L4 nematodes into a new plate.
2. Day 2: Wait.
3. Day 3: Transfer all L4 nematodes from the original plate to a new plate, also called Day 2 L4.
4. Wait for 4 hours until the L4 nematodes lay eggs, and remove the parent worms.
5. Day 4: Wait
6. Day 5: Wait
7. Day 6: After incubating for 60 hours, the plate will be filled with L4 nematodes. Wait one night.
8. Day 7: At this point, the plate is filled with Day 1 L4 nematodes, which can be directly used in chemotaxis experiment.

**Material:**

1. Caenorhabditis elegans, E.coli OP50, nematode growth medium
2. Take 1ml of the initial algal solution in a super clean table
3. The algal liquid was diluted about 100 times by gradient dilution method

#### Chemotaxsis

![experiment-chemotaxsis](https://static.igem.wiki/teams/4900/wiki/experiment/experiment-chemotaxsis.jpg)

**The definition and operation method of two-way selection experiment:**

1. Two-way selection experiment is an experiment to reveal the tendency of matter to nematode. Two-way selection experiments use a confined space with sample and control areas and a large number of nematodes.
2. For chemicals with strong volatility, a larger space is generally used for experiments. In our iGEM experiment,algal liquid is used, which is less volatile, so a T-shaped mold is used instead to limit the activity range of nematodes. There were three areas in the experimental area: sample, control and initial location of nematode. The results were obtained by observing the distribution of nematode and calculating the chemotactic index i. For each individual experimental area, it is recommended to add 100 nematodes to ensure accurate experimental results.

**Operation method:**

1. Prepare a large petri dish with palmitic acid crystals \*\* in advance. To obtain sufficient amount of D1 nematode using synchronization, add 1mL S-buffer\* to the petri dish with nematode, shake and rotate the medium to soak the surface nematode into the buffer, tilt the medium to suck out the buffer with nematode and continue to add it to the next medium. Repeat the above operations to collect nematode. Because the medium absorbs liquid, it can be properly supplemented when the liquid is insufficient, and if there are too many nematodes in the liquid, the liquid can be collected into the centrifuge tube first, and the S-buffer can be re-taken to collect other nematodes. buffer collection can be added multiple times to reduce nematodes remaining on the medium and reduce losses. After all nematodes were collected in the centrifuge tube, the nematodes were allowed to sit for 3 minutes until the nematodes sank to the bottom, the upper liquid was sucked out, and an appropriate amount of S-buffer was added again to wash the nematodes. After standing for 3 minutes, the above operations were repeated for 3 times, and finally the upper liquid was washed again with distilled water. After standing, the upper liquid was sucked out, leaving about 300 microliters of liquid for use. The whole process is best completed within 30min, otherwise it may reduce the vitality of nematode. The role of washing is to remove E. coli food from the liquid and prevent food interference.
2. After the crystallization of palmitic acid is completed, 50 microliters of sample (algal liquid or algal liquid diluted in medium) and control (algal medium) are added to the left and right sides respectively. when the nematode totally sink to the bottom ,the sampling operation can start. After the added liquid is absorbed, the nematodes can be added to the experimental dish. Nematodes were added under the T-shape in batches of 5 microliters at a time with a 10 microliter pipette gun. The nematodes added in each petri dish were observed and estimated, and the nematodes added in each medium were reasonably distributed to achieve a stable average as far as possible. After adding nematodes, use absorbent paper to carefully blot away excess liquid until nematodes can move normally on the medium. If the nematode is normally active, the lid can be covered, and counting can be done after 30min. The distribution range of nematode in each area can be counted, and the chemotactic index \*\*\* can be calculated and the results obtained.

#### Protocol for construction

![experiment-plasmid](https://static.igem.wiki/teams/4900/wiki/experiment/experiment-plasmid.jpg)

### A.Construction

**1.Purpose**

1. Make *C. elegans* expresses β-galactosidase under stress state, and then β-galactosidase can catalyze X-Gal substrate (β-galactosidase chromogenic substrate) to turn blue. In that case, the field detection is more convenient.

**2.Principle**

1. Original plasmid: Pbbs-1: TIR: SL2: mcherry
2. Target plasmid: Phsp-6: Met+SV40 NLS: LacZ: SL2: mcherry
3. Phsp-6: Mitochondrial stress promoter, it may respond to increased oxygen free radical (ROS) levels caused by microcystin (MC-LR)
4. Met+SV40 NLS: Can guide LacZ gene into the nucleus
5. LacZ: Code for β-galactosidase
6. SL2: Trans-splice
7. Mcherry: Red fluorescent protein, which is used to observe whether the plasmid penetrates into the worm successfully

**3.Process**

1. Replacement of the promoter: Pbbs-1→Phsp-6
2. Replacement of the gene: TIR→LacZ
3. Ⅲ. Insert SV40 NLS

### B.Fluorescence photography

1. The nematodes used in the experiment were treated synchronously to reduce the difference of nematodes, and the nematodes of L4 period were selected as needed
2. Prepare medium with different concentrations of MC-LR (mixed with OP50 bacterial solution), and mark the basic information such as the concentration date
3. The L4 stage nematodes selected in the first step were placed in petri dishes with different concentrations, and the placement time was recorded
4. Prepare a 2% concentration of agarose plate
5. Place nematodes on sterile plates with eyelash pickers (remove bacterial liquid on the surface of nematodes), and then place them on prepared plates with eyelash pickers (eyelash pickers prevent nematode stress).
6. The plate with nematodes arranged was placed under a fluorescence microscope, and relevant software was opened to observe nematodes under bright field and blue light source (exposure time 500ms, excitation light wavelength 488nm), and the nematodes in different states were photographed
7. Use software to analyze fluorescence differences and make charts

[Learn more](https://static.igem.wiki/teams/4900/wiki/file/experimental-details.pdf)

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{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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[

Your browser does not support the video tag.

# Left side:solution with algal liquid

# Right side:control group

](https://video.igem.org/download/streaming-playlists/hls/videos/b1eb37a3-98e7-4eb1-b09e-b388eecc1212-720-fragmented.mp4)

#### Result 1 Chemotaxsis assay

**1.Two-choice preference assays experimental record/procedure**

**Preparation of two-choice preference assay plates:**

1. Take a 1L conical bottle and wash it
2. We obtained a specific concentration of algal suspension, filtrated a portion of it, and took the culture medium as a control for two-choice preference assay. t
3. We designed a T-type experimental area on the plates and pleaced 100µl of each inoculum diagonally opposite onto assay plates.

**The testing procedure of the two-choice preference assay:**

1. We recorded the movement of *C. elegans* on the two-choice preference assay plates by video recording.
2. We first explored the choice bias of *C. elegans* when the concentration of algal suspension is 10^7 cells /L.
3. We observed that *C. elegans* moved back and forward between the algal suspension and the control culture medium at the beginning of the experiment.

A few minutes later, we observed a significant difference in the number of *C. elegans* on the algal suspension and on the control culture medium.

Later in the experiment, most of the *C. elegans* on the control culture medium leaving and approaching the algal suspension were observed. But most of the *C. elegans* on the algal suspension remained in this region. The result showed that the number of C. elegans on the algal suspension was much greater than that on the control culture medium. After two-choice preference assay using different concentrations of algal suspension, We found that the minimum/boundary concentration of algal suspension for the formation of C. elegans choice bias was 2\*10^6 Cell/L.

**2.Data analysis of two-choice preference assays**

1. The chemotaxis index (i) is an important index for the analysis of the results of the two selection experiments.
2. i=[(No.of worms on Liquid of algae)-(No.of worms on culture medium)]/Total number of worms on plates
3. Generally, when the chemotaxis index is greater than 0, it indicates that  *prefers algal suspension to culture medium. The closer the chemotactic index is to 1, the more attractive the algal fluid is to nematodes.*
4. In the actual experiment, if the average chemotactic index of the same concentration is greater than 0.5, it can be shown that the algal suspension is more attractive to *C. elegans* than the culture medium.

![result-fig1](https://static.igem.wiki/teams/4900/wiki/result/result-fig1.png)

Figure 1. Responses of Wild Type to Different Concentrations of Microcystis aeruginosa
Unoperated wild-type animals weretested for their responses to Microcystis aeruginosa.
Chemotaxis index =(animals at the Microcystis aeruginosa -animals at the control)/total animals on the plate. A chemotaxis index of 1.0 indicates complete attraction; a chemotaxis index of
-1.0 indicates complete repulsion. Each column represents the average of three independent chemotaxis assays, using ~100 animals in each assay. Error bars equal the SEM. This result has been repeated ten times and each repeat shows similar tendency.

* Based on the results of two-choice preference assay(Figure 1), which is *C. elegans* preferred algal suspension, indicating that Microcystis aeruginosa can attract *C.elegans* and influence its behavior.
* *C. elegans* have the ability to detect whether the water contains Microcysts aeruginosa.
* When the concentration of algal suspension was 10^7 cells /L, the chemotactic index was about 0.6,which was the highest value under all experimental conditions.
* The chemotaxis index decrease slowly while the concentration decrease.
* The results showed that the index is still around 0.5 when the concentration of algal suspension is 2\*10^6 cell/L, until the concentration is 10^6 cell/L, there is a sudden drop of index, finally at the 10^5 cell/L, *C.elegans* can hardly sense the existence of Microcystis aeruginosa(Figure 1).
* According to the standard [1], it defines the "no significant water blooming" concentration is 2\*10^6 to 10^7cell/L, and the sudden drop of the index overlap with this standard concentration. So our device has the expected function to detect the water blooming before it really happens and leads to environment damage.
* As the same time, our experimental data showed that the locomotor behavior ofi *C. elegans* can be observed without relying on the microscope, which indicated that our device have the potential to shrink the size and perform the real-time examine at the sampling places without the microscope.

#### Result Ⅱ Color Reaction Assay

![result-fig1](https://static.igem.wiki/teams/4900/wiki/result/result-fig2.png)

Figure 2. Fluorescence Images of the Phsp-6:GFP Reporter Strain after MC-LR Treatment
(A) Brightfield and fluorescence images of the Phsp-6:GFP reporter strain after conventional culture and MC-LR treatment. Animals were moved to control or testing plate at L4 stage, the photos were taken after 24h treatment.
(B) Quantification of Phsp-6:GFP fluorescence intensity (n ≥ 15; n is the number of biological replicates; Mean is shown by bar; \*\*\*p is smaller than 0.0001 by two-tailed t-test).

However, various organisms are contained in natural water bodies, and we need to further confirm that Microcystis aeruginosa plays a leading role in worm attracting. Microcystis aeruginosa secrets microcystin LR (MC-LR). It has been reported that MC-LR increases ROS levels in C.elegans, while ROS induces mitochondrial unfolded protein response (UPRmt). Accordingly, we designed our second step assay, using Phsp-6, a promoter specifically activated by UPRmt, to confirm whether MC-LR induces UPRmt and increases expression of reporter genes downstream of Phsp-6. We treated Phsp-6:GFP transgenic strain with 100 μg/L MC-LR for 24h and found that, compared to the control, the fluorescence intensity significantly increased (Figure 2), indicating that MC-LR treatment activated UPRmt and induced high expression of GFP.
In summary, as our two-step experiments have shown, worms are attracted by algal suspension, while MC-LR activates UPRmt of nematodes. These results strongly suggest that there may be high concentrations of Microcystis aeruginosa in the water, which may lead to water blooms.
In order to detect the UPRmt level of worms in a convenient assay, independent on expensive and unportable fluorescence microscopes, we replaced GFP with lacZ to construct the plasmid Phsp-6:SV40 NLS:lacZ:SL2:mcherry (Figure 3). lacZ is a bacteria-derived gene, encoding β-Galactosidase, which transforms X-gal substrate to blue insoluble precipitate. This obvious discoloration can be easily observed by naked eyes. We provides a method for easier detection of UPRmt activation level through color reactions (data not shown).
![result-fig1](https://static.igem.wiki/teams/4900/wiki/result/result-fig3.png)

Figure 3. A Schematic of Plasmid.

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