Team:BNU-CHINA/Results

Team:BNU-CHINA - 2015.igem.org

l-/d-limonene synthase

Agarose gel electrophoresis of l-/d- limonene synthase gene

In Fig.1 limonene synthase gene is inserted into the backbone pSB1C3. Then the plasmids are digested by Pst I and EcoR I. The length of the l-LS gene, d-LS gene and backbone are 1735bp, 1904bp and 2624bp,respectively. The target genes are marked with arrows, and the figure shows that limonene synthase gene was transferred into the E.coli BL21 cells successfully. 1.2 SDS-PAGE analysis of l-/d- limonene synthase

SDS-PAGE analysis of l-/d- limonene synthase

Fig.2 SDS-PAGE of l-limonenen synthase, d-limonene synthase and Gpps Lane M, molecular weight standards (kDa); Lane2 and 3, bacteria homogenate of d-limonene synthase; Lane 4 and 5, bacteria homogenate of l-limonene synthase; the lower bands pointed by arrows show GPPS and the upper bands pointer by arrows show limonene synthases.

In Fig 2，we want to verify whether the limonene synthase and GPPS gene are expressed or not. The molecular weight of limonene and GPP synthase is 89 kDa (with GST on pGEX-4T-1 plasmid) and 49 kDa respectively. In Fig.2, the target proteins are marked with arrows, and the figure shows that synthases are expressed in the cell successfully.

Verification of attraction of limonene towards C. elegans

In order to know whether limonene attracts C. elegans, filter paper is dropped of 5 $\mu$L5% limonene (DMSO aq) and put on one side. The control is put on the other side with only 5 $\mu$L DMSO. To eliminate the effect of DMSO, two other control groups are made.

Table 1. The results of verification of limonene to attract C. elegans

		1	2	3
Control guoup 1	DMSO	117	113
	DMSO	110	159
Control group 2	DMSO	180	86	30
	M9	198	91	26

a. The distribution of the C. elegans on the plates of control group Control group is used to eliminate the effect of DMSO, and some other experimental factors.

	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
DMSO	110	8	61	129	74	90	19	51	172	144	89	125	49	114	28	63	109	346	54	70
Limonene+DMSO	149	64	37	185	128	100	94	150	202	190	114	124	43	129	57	101	261	495	59	79

b. The distribution of the C. elegans on the plates of experimental group

This group shows significantly biased movement by compareing l-limonene with DMSO.

We find that nematodes of experimental group show significantly biased movement when we count the number of nematodes (Fig.1). However, nematodes of control group shows no significant difference about the distribution of nematodes on the plate.

Fig.1 The distribution of the C. elegans on the plates.

C stands for the control group in which the filter paper is dropped with 5 $\mu$L DMSO; T stands for the experimental group in which the filter paper is dropped with 5 $\mu$L 5% l-limonene.

According to the data we get, we did simple analysis which means we used the ratio of the number of nematodes distributed on two sides and drew a histogram(Fig.2). In the histogram, nematode distribution of experimental group shows significant difference that nematodes prefer limonene.

Fig2. Proportion of nematodes’ distribution

During our verification of attraction of limonene, we analyzed 20 samples. In order to verify if there is any difference between test group and the control group at statistic level, we used paired t test to verify.

The methods are shown as the following:

The confidence of the following is 0.05, α=0.05. Firstly, we used the normality test. We selected the single sample K-S test to verify whether it had normality.

Judging standard of the single sample K-S test
If P > 0.05, it has normality, otherwise it doesn’t have normality.
We used SPSS to derive the data and output the results

Table.1 the output of the normality test

	Control	Test
Sample size	19	19
Mean	97.0526	143.3684
Variance	74.46362	102.14108
K-S statistics	0.767	0.920
P value	0.598	0.366

Analysis

The p value of the control group is 0.598, while that of the test group is 0.366. Both of these two groups meet normality.

After testing the normality, we did a significance test——paired t test to verify if there was any difference between these two groups.

1. Set up a hypothesis

$$H_0: \mu1 = \mu2$$

$$H1: \mu1 < \mu2$$

Among them, $\mu_1$ is the mean of the control group, $\mu_2$ is the mean of the test group.

2. The judging standard of the paired t test.

If p < 0.05, we reject H₀ and accept H₁ and the limonene can attract the nematodes. And if p > = 0.05, we accept H₀ and the limonene has no effect on the nematodes.