Section 1 Introduction
In 2023, our project aims to build an “Algalchip” tokkkkkkk detect the concentration of algae and make early warning towards water bloom.
Building our Algalchip requires two steps:
- Training C. elegans to only move to the sample which meets or above prewarning concentration of algae.
- Modifying C. elegans to make it sensitive to microcystin.
Section 2 Parts
The parts are essential to gene expression. Our project has registered multiple parts, all qualified for standardized requirement of RFC[10].
- Phsp-6 promoter
Phsp-6 is the mitochondrial stress promoter, it can respond to increased oxygen free radical (ROS) levels caused by external stimulus like the existence of microcystin (MC-LR). In that case, it could be used to reflect the change or stimulation of the surroundings.
- LacZ
The LacZ can code for β-galactosidase inside the C. elegans under stress state. We made a few point mutations to optimize codon usage, to make sure that the LacZ can be spliced normally and correctly in the C. elegans. The β-galactosidase can catalyze X-Gal substrate to turn blue. In that case, we can detect the change of the surroundings without the help of the fluorescence microscope.
Section 3 Experience of wet lab
- C.elegans treatment
- Unable to correctly identify forth stage larva (L4) C. elegans
For our experiment, we need to identify the C. elegans in the culture medium at a specific stage, which is the L4 stage. At this stage the C. elegans have a gonadal syncytium which shaped like an oval cavity located near the 1/3 of one end of C. elegans. But quickly distinguishing this gonadal syncytium among hundreds of nematodes on a culture medium under a microscope was not an easy task. At the beginning we often picked the wrong C. elegans, which prevented us from carrying out our experiments properly. But after we practiced for a period of time, we were able to correctly and accurately distinguish the C. elegans of the right period.
- Unable to control the strength
We also have to control the strength when picking the C. elegans, we can't scratch the culture medium otherwise the C. elegans will burrow into the gaps of the medium and can't be killed during the synchronization process. It will then produce offspring in the stage different from others, affecting our synchronization results. Also, C. elegans are fragile so if we cannot control our strength, they might get hurt when picking.
- Bacterial contamination
When picking C. elegans, the culture medium often comes into contact with the air, which can lead to the culture medium being infected by fungi or bacteria. In order to prevent the infection of bacteria, we have taken various measures, such as wearing lab coats and gloves during the experiment, disinfecting the experimental area and our body using alcohol before the experiment, opening the lid of the medium as little as possible when transferring the C. elegans and speeding up the transfer to reduce the contact of the culture medium with the air.
- Chemotaxis
We are trying to find the chemotaxis of C.elegans between culture solution and solution contained algae. The concentration gradient should be decided carefully and before experiment we need to do the pre-experiment. Besides, the observation time should not be long, about 1 hour and 15 minutes, and the proportion of nematodes climbing out of the experimental area is high. So the best observed time is about 45 minutes after the drip. When printing palmitic acid, try to make the border obvious to avoid nematodes climbing out. For each time, we need to control the number of nematodes. Too many nematodes can lead to a lack of space, and many nematodes are in the middle and hard to count. Less nematodes are not enough to prove if there is a chemotaxis.
- Algal liquid
Due to the lack of systematic understanding of algae, the team encountered great difficulties in the preservation of algal solution, and it was difficult to maintain the stability of algal solution concentration and purity of algal solution. At the same time, it was also difficult to measure the concentration of algal liquid. We initially chose the method of microscopic inspection through literature reading, but it was difficult to carry out conveniently and effectively due to the limitation of manpower and instruments. The above two points have been effectively solved after contact and communication with the teachers of the aquatic Institute.
Section 4 Hardware
Our hardware is an “Algalchip” which can be used to detect the concentration of algae and make early warning towards water bloom. Our “Algalchip” has two main duties. Once the water sample is dipped into the sample region, firstly the C. elegans in the our “Algalchip” can perform chemotaxis essay. It can be used as a simple measurement for whether the concentration of algae in the water sample has reached the prewarning concentration. Secondly, modified C. elegans in “Algalchip” are able to turn blue when they were exposed to prewarning concentration and above of microcystin. The second part serves as a double check of our detecting results, making sure that it is the cyanobacteria that attracts C. elegans to move towards the sample. In general， our product are aimed to offer a real-time, rapid, non-toxic, and affordable bio-detector for the ecological environment in lakes, ultimately ensuring the safety of our aquatic food products.