Difference between revisions of "Team:Hong Kong UCCKE/Experiments"
| Line 43: | Line 43: | ||
<div class="col-sm-9 col-xs-12"> | <div class="col-sm-9 col-xs-12"> | ||
<h2>Experiments</h2> | <h2>Experiments</h2> | ||
| − | <h3>Assay</h3> | + | <h3 style="margin-top:0;">Assay</h3> |
| − | <p | + | <p style="text-align:left; font-size:15px;"> |
| − | < | + | We have done assays on pure chemicals and bacteria synthesized chemicals. |
| − | + | <br> | |
| + | 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. | ||
| + | <br> | ||
| + | First, as shown in the following image, we mark the substance loading spot on the lid where we put volatile substances. | ||
| + | <br> | ||
| + | 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. | ||
| + | <br> | ||
| + | After labelling, we load 10 ul of medium containing about 200 worms on the centre of the the agar. | ||
| + | <br> | ||
| + | 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. | ||
| + | <br> | ||
| + | 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. | ||
| + | <br> | ||
| + | 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. | ||
| + | <br> | ||
| + | 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. | ||
| + | <br> | ||
| + | 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. | ||
| + | <br> | ||
| + | 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. | ||
| + | <br> | ||
| + | We tested with pure chemicals of cinnamaldehyde and phenylpyruvic acid to check if our assumptions about the attractiveness were correct. | ||
| + | <br> | ||
| + | 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. | ||
| + | <br> | ||
| + | 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. | ||
| + | </p> | ||
</div> | </div> | ||
<div class="col-sm-3 col-xs-12"> | <div class="col-sm-3 col-xs-12"> | ||
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<div class="col-sm-9 col-xs-12"> | <div class="col-sm-9 col-xs-12"> | ||
<h2>Protocols</h2> | <h2>Protocols</h2> | ||
| − | <h3>3A Assembly</h3> | + | <h3 style="margin-top:0;">3A Assembly</h3> |
<p><a href="https://static.igem.org/mediawiki/2016/9/9c/T--Hong_Kong_UCCKE--3aassemblylabmenu.pdf" download="3A_Assembly_Protocol">Download <i class="fa fa-download" aria-hidden="true"></i></a></p> | <p><a href="https://static.igem.org/mediawiki/2016/9/9c/T--Hong_Kong_UCCKE--3aassemblylabmenu.pdf" download="3A_Assembly_Protocol">Download <i class="fa fa-download" aria-hidden="true"></i></a></p> | ||
<embed src="https://static.igem.org/mediawiki/2016/9/9c/T--Hong_Kong_UCCKE--3aassemblylabmenu.pdf" type="application/pdf" width="100%" height="650px"> | <embed src="https://static.igem.org/mediawiki/2016/9/9c/T--Hong_Kong_UCCKE--3aassemblylabmenu.pdf" type="application/pdf" width="100%" height="650px"> | ||
Revision as of 01:08, 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.
