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Revision as of 01:18, 1 October 2016
Experiments
Assay
We have done assays on pure chemicals and bacteria synthesized chemicals.
The aim of doing these assays was to test whether the substances are attractant or repellent to C. elegans. And we use a response index to describe whether the chemical is an attractant or repellent to C. elegans. Here are the steps and calculations in detail.
First, as shown in the following image, we mark the substance loading spot on the lid where we put volatile substances.
Then mark the same with an extra circle around each loading spot. Label two opposite area as ‘T’ and the other two as ‘C’. they stand for ‘test’ and ‘control’ respectively.
After labelling, we load 10 ul of medium containing about 200 worms on the centre of the the agar.
For volatile test, we pipetted 2 ul of tested volatile substance on the loading spot in the two ‘T’ areas on the lid. And load the same volume of water on the ‘C’ spots on the lid.
For droplet test, we pipetted 5ul of tested liquid on the loading spot in the two ‘T’ areas on the agar plate. And load the same volume of water on the ‘C’ spots on agar too.
Then, we put the assay plate aside and leave it undisturbed for 30 minutes. After 30 minutes, we obtained the total number of worms in the ‘T’ areas and ‘C’ areas.
The response index was calculated by formula “ T minus C over T plus C“ . T is the number of worms in the ‘T’ areas. And C is the number of worms in the ‘C’ areas, which means the number of worms that entered the areas by chance . The denominator ‘T plus C’ represents the total number of worms in the counted areas, that is inside the 4 circles. The numerator T minus C is the number of worms which made response due to the effect of the tested substances.
The index value will range between -1 to 1. If the index is negative, it means the tested chemical is repellent; in contrast, positive index means the tested chemical is attractant.
We tested our assay and response index by chemical diacetyl, a known strong attractant to C. elegans. And the result is promising that the Response index of volatile is close to 1.0, which means that assay and response index can show diacetyl is a strong attractant. We also tried using water as negative control for the assay. The Response index is 0.0x. Which means the assay can show that the worms has no significant response, neither attraction or repulsion, to water.
We tested with pure chemicals of cinnamaldehyde and phenylpyruvic acid to check if our assumptions about the attractiveness were correct.
The result is that index of cinnamaldehyde is -0.81 when concentration is 1, -0.8 at 1/10, -0.82 at 1/100, -0.5 at 1/1000, -0.13 at 1/10000 and -0.1 at 1/100000. Phenylpyruvic acid’s index is 0.34 when concentration is 1, 0.31 at 1/10, 0.28 at 1/100, 0.18 at 1/1000, 0.04 at 1/10000 and -0.01 at 1/100000. With the evidence provided by the tests, the results are clearly shown. Cinnamaldehyde is a strong repellent and Phenylpyruvic acid is a moderate attractant. Please refer to the result page for a more detailed assay result.
After testing pure chemicals of cinnamaldehyde and phenylpyruvic acid, we have to do a similar assay on bacteria synthesized cinnamaldehyde and phenylpyruvic acid. Other than testing the bacteria synthesized chemicals, we also conducted assay to test on bacteria containing backbone-only plasmid as a negative control with water remaining as a control. This is to ensure it is the bacteria synthesized chemical which affect the assay result instead of the plasmid or bacteria itselves involving in the results. The response indexes taken in assay on bacteria synthesized phenylpyruvic acid and backbone-only plasmid bacteria are 0.83 and 0.63 respectively. The result indicates that the plasmid and bacteria itself is already an attractant to C.elegans. However, phenylpyruvic acid can be produced by the bacteria and had increased the overall attractiveness. For cinnamaldehyde, we were not able to clone the genes into a plasmid and do transformation and thus we did not conduct any assay on bacteria synthesized cinnamaldehyde.
Cloning
Based on the assays done on pure chemicals, cinnamaldehyde is a repellent while phenylpyruvic acid is an attractant. Hence, it would be more convenient for us to choose parts that are registered on the iGEM. We found a synthesis pathway of cinnamaldehyde with genes in the iGEM parts library, given by the 2013 UBC team. The pathway is constructed with 3 genes, where the starting substrate is phenylalanine, an amino acid. With the sequence of the three genes provided, we designed and ordered the 3 oligo DNA from IDT, the Integrated DNA Technologies, Inc..
Each ordered oligo DNA sequence encloses a constitutive promoter J23100, medium strength RBS B0034, a gene in the cinnamaldehyde synthesis pathway and a double terminator, with iGEM prefix and suffix for a more convenient use in 3A assembly cloning steps.
We were supposed to subclone these genes into 3 separate plasmid backbones then clone the 3 parts into one plasmid backbone. And lastly transform the plasmid into bacteria. Unfortunately, we were not successful in doing it.
Phenylpyruvic acid is an attractant. However, we had difficulties find a protein sequence that enables the production of phenylpyruvic acid in the iGEM community. As such, we decided to synthesize it ourselves. We discovered a protein sequence for producing phenylpyruvic acid on NCBI website. We again ordered the oligo DNA from IDT. The ordered sequence also encloses a constitutive promoter J23100, medium strength RBS B0034, the target gene and a double terminator, with iGEM prefix and suffix. Our new gene is an amino acid deaminase gene.
So we’ll use this composite part to perform our assays.
