For our experiments, we did 2 types of volatile tests: one for diacetyl and another for cinnamaldehyde.

Positive Control for Attractant

For the diacetyl volatile test, 6 sets of the tests were performed using different concentrations of diacetyl. Each set was repeated twice to obtain an average response index to maximize accuracy. For 1x concentration of diacetyl, the average index was +0.96. At 1/10x concentration, the average index dropped slightly to +0.84. From then on, it dropped dramatically to +0.27 at 1/100x concentration, then to +0.17 at 1/1000x concentration. At 1/10000x concentration, the index was at +0.05. But it rose slightly to +0.06 at 1/100000x concentration.

All the response indices collected were positive. This suggests that diacetyl is a volatile attractant. The high index recorded at 1x concentration shows that diacetyl is the most potent attractant. However, as evidenced by data from 1x concentration to 1/1000x concentration, when concentration is lowered, its potency as an attractant decreases. It can be concluded, hence, that the potency of diacetyl as an attractant is directly proportionate to its concentration up till 1/1000x concentration. The slight rise in average index at 1/100000x concentration suggests that 1/100000x diacetyl is a slightly stronger attractant than 1/10000x diacetyl.

As a result, this assay proves that pure diacetyl attracts C. elegans and can be utilized in our experiments as a positive control for attractant.

Chemical	Type	Concentration	# of worms in T	# of worms in C	Index
Diacetyl	Volatile Test	1	10	0	1
Diacetyl	Volatile Test	1	80	3	0.927710843
Diacetyl	Volatile Test	1	96	2	0.959183673
Diacetyl	Volatile Test	1/10	97	8	0.847619048
Diacetyl	Volatile Test	1/10	78	9	0.793103448
Diacetyl	Volatile Test	1/10	91	6	0.87628866
Diacetyl	Volatile Test	1/100	7	4	0.272727273
Diacetyl	Volatile Test	1/100	85	54	0.223021583
Diacetyl	Volatile Test	1/100	60	32	0.304347826
Diacetyl	Volatile Test	1/1000	18	14	0.125
Diacetyl	Volatile Test	1/1000	67	45	0.196428571
Diacetyl	Volatile Test	1/1000	51	35	0.186046512
Diacetyl	Volatile Test	1/10000	13	13	0
Diacetyl	Volatile Test	1/10000	27	22	0.102040816
Diacetyl	Volatile Test	1/10000	27	24	0.058823529
Diacetyl	Volatile Test	1/100000	18	16	0.058823529
Diacetyl	Volatile Test	1/100000	32	29	0.049180328
Diacetyl	Volatile Test	1/100000	35	31	0.060606061

Negative control

The average response index of water is taken as +0.07, obtained among 6 repeated assays. This indicates water is a neutral chemical towards C.elegans. considering this, we use water as a negative control for onwards experiments.

CA

For the cinnamaldehyde volatile test, 6 sets of the tests were performed using different concentrations of cinnamaldehyde. Each set was repeated thrice this time. The results were processed to produce an average response index. For 1x concentration of cinnamaldehyde, an average index of -0.81 was recorded. The index then fluctuates slightly to -0.80 at 1/10x concentration and to -0.82 at 1/100x concentration subsequently. The index then drops to -0.50 at 1/1000x concentration. The index decreases dramatically to -0.13 at 1/100000x concentration. It finally decreases to -0.10 at 1/10000x concentration.

All the indices collected were negative. This suggests that cinnamaldehyde is a volatile repellant. The similarly high negative indices at 1x concentration to 1/100x concentration show that cinnamaldehyde is a strong repellant. The difference in indices is only 0.01, hence, it suggests that the differing concentrations have little to do with the repelling effect of cinnamaldehyde. The negative indices then decrease along with concentration as evidenced by the data from 1/1000x concentration 1/100000x concentration. This shows that the repelling effect is directly proportionate to the concentration of the cinnamaldehyde.

As a result, the assay proves that pure cinnamaldehyde repels C. elegans and can be used for our experiments.

Chemical	Type	Concentration	# of worms in T	# of worms in C	Index
Cinnemoaldehyde	Volatile Test	1	18	75	-0.612903226
Cinnemoaldehyde	Volatile Test	1	0	24	-1
Cinnemoaldehyde	Volatile Test	1	11	92	-0.786407767
Cinnemoaldehyde	Volatile Test	1	9	95	-0.826923077
Cinnemoaldehyde	Volatile Test	1/10	10	97	-0.813084112
Cinnemoaldehyde	Volatile Test	1/10	3	32	-0.828571429
Cinnemoaldehyde	Volatile Test	1/10	13	93	-0.754716981
Cinnemoaldehyde	Volatile Test	1/10	11	98	-0.798165138
Cinnemoaldehyde	Volatile Test	1/100	6	90	-0.875
Cinnemoaldehyde	Volatile Test	1/100	2	38	-0.9
Cinnemoaldehyde	Volatile Test	1/100	9	68	-0.766233766
Cinnemoaldehyde	Volatile Test	1/100	14	96	-0.745454545
Cinnemoaldehyde	Volatile Test	1/1000	27	82	-0.504587156
Cinnemoaldehyde	Volatile Test	1/1000	9	28	-0.513513514
Cinnemoaldehyde	Volatile Test	1/1000	14	40	-0.481481481
Cinnemoaldehyde	Volatile Test	1/1000	17	50	-0.492537313
Cinnemoaldehyde	Volatile Test	1/10000	40	35	0.066666667
Cinnemoaldehyde	Volatile Test	1/10000	16	28	-0.272727273
Cinnemoaldehyde	Volatile Test	1/10000	27	37	-0.15625
Cinnemoaldehyde	Volatile Test	1/10000	29	39	-0.147058824
Cinnemoaldehyde	Volatile Test	1/100000	34	44	-0.128205128
Cinnemoaldehyde	Volatile Test	1/100000	25	37	-0.193548387
Cinnemoaldehyde	Volatile Test	1/100000	28	33	-0.081967213
Cinnemoaldehyde	Volatile Test	1/100000	32	33	-0.015384615

Phenylpyruvic Acid

For the phenylpyruvic acid droplet test, 6 sets of the tests were performed using different concentrations of chemical ranging from 1x to 1/100000x. Each set was repeated thrice for a constant result. The results were processed to produce an average responsive index. For 1x concentration of phenylpyruvic acid, an average index of +0.34 was recorded. The index then declines slightly to +0.31 at 1/10x concentration and to +0.28 at 1/100x concentration subsequently. The index then drops to +0.18 at 1/1000x concentration. The index further decreases to +0.04 at 1/100000x concentration. It finally decreases to -0.01 at 1/10000x concentration.

All response index from the phenylpyruvic acid droplet tests are positive except at concentration 1/100000x with index of -0.01. The indices obtained at concentrations 1/1000x, 1/10000x and 1/100000x are too close to 0, that is a neutral substance. Therefore the data obtained from the three lowest concentrations in this test is ignored and cannot give a conclusion. As the response indices at 1x, 1/10x and 1/100 concentration phenylpyruvic acid are positive and are indicate a relatively significant attractiveness of C.elegans, we would suggest phenylpyruvic acid is a mild attractant to C.elegans. Also, at the above concentrations, the indices decreases consequently with intervals 0.03 and then drop significantly onwards. Therefore we have to use concentrations between 1x and 1/100x of phenylpyruvic acid in further experiments to obtain meaningful results. The indices decrease as the concentration of phenylpyruvic acid decrease, showing the attracting effect is directly proportional to the concentration of phenylpyruvic acid.

To conclude, this assay proves that pure phenylpyruvic acid attracts C. elegans and can be used for our experiments.

Chemical	Type	Concentration	# of worms in T	# of worms in C	Index
Phenylpyruvic Acid	Droplet Test	1	108	45	0.411764706
Phenylpyruvic Acid	Droplet Test	1	14	8	0.272727273
Phenylpyruvic Acid	Droplet Test	1	76	42	0.288135593
Phenylpyruvic Acid	Droplet Test	1	63	27	0.4
Phenylpyruvic Acid	Droplet Test	1/10	15	9	0.25
Phenylpyruvic Acid	Droplet Test	1/10	17	11	0.214285714
Phenylpyruvic Acid	Droplet Test	1/10	63	29	0.369565217
Phenylpyruvic Acid	Droplet Test	1/10	55	23	0.41025641
Phenylpyruvic Acid	Droplet Test	1/100	106	56	0.308641975
Phenylpyruvic Acid	Droplet Test	1/100	15	11	0.153846154
Phenylpyruvic Acid	Droplet Test	1/100	43	23	0.303030303
Phenylpyruvic Acid	Droplet Test	1/100	33	16	0.346938776
Phenylpyruvic Acid	Droplet Test	1/1000	50	34	0.19047619
Phenylpyruvic Acid	Droplet Test	1/1000	12	7	0.263157895
Phenylpyruvic Acid	Droplet Test	1/1000	47	36	0.13253012
Phenylpyruvic Acid	Droplet Test	1/1000	23	18	0.12195122
Phenylpyruvic Acid	Droplet Test	1/10000	70	64	0.044776119
Phenylpyruvic Acid	Droplet Test	1/10000	11	9	0.1
Phenylpyruvic Acid	Droplet Test	1/10000	25	23	0.041666667
Phenylpyruvic Acid	Droplet Test	1/10000	37	38	-0.013333333
Phenylpyruvic Acid	Droplet Test	1/100000	53	57	-0.036363636
Phenylpyruvic Acid	Droplet Test	1/100000	36	35	0.014084507
Phenylpyruvic Acid	Droplet Test	1/100000	25	28	-0.056603774
Phenylpyruvic Acid	Droplet Test	1/100000	19	18	0.027027027

Bacteria synthesized chemicals

We are only able to clone plasmid with phenylpyruvic acid production enabling gene. We used transformed bacteria containing the composite part to produce synthetic PA. The assay is being repeated twice. Its average response index was +0.83. Comparatively, the average index of transformed bacteria with backbone-only plasmid was +0.63, while it is known that bacteria attracts C. elegans as a food source, the data proves that our bacteria indeed has potency as an attractant that is independent of its nature as a food source. Hence, our bacteria are attractants to C. elegans in themselves. The transformed bacteria had a higher index than PA, suggesting that the bacteria are stronger attractants. However, the bacteria still have a lower index than diacetyl.

Chemical	Type	# of worms in T	# of worms in C	Index
Synthetic PA	Droplet Test	62	7	0.797101449
Synthetic PA	Droplet Test	352	27	0.857519789
Synthetic PA	Droplet Test	82	7	0.842696629
Empty Vector	Droplet Test	47	6	0.773584906
Empty Vector	Droplet Test	55	12	0.641791045
Empty Vector	Droplet Test	75	18	0.612903226
Empty Vector	Droplet Test	47	16	0.492063492

Overall Comparison

As an overall comparison of pure chemicals, the response index for diacetyl was 0.96, which is tested to be a strong attractant and has been used as a positive control for attractant. Water was used as a negative control and obtained an index of +0.07. Phenylpyruvic acid, having the lowest positive responsive index of +0.34 is suggested to be a mild attractant. On the other hand, pure cinnamaldehyde had -0.81 response index. It was the only chemical that had a negative index, suggesting that it is the only repellant in our range of chemicals. It is a strong repellant as evidence with its obtaining a highly negative index. The index obtained from the bacteria synthesized PA droplet test is higher than that obtained from backbone-only plasmid bacteria. This suggests the transformed bacteria has performed our desired effect.