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

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

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.

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

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