Team:BNU-CHINA/Results

Project Results

l-/d-limonene synthase

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

Fig.1 the agarose gel electrophoresis of l-/d- limonene synthase gene, the bands pointed by arrows represent l-limonene gene and d-limonene gene. Lane M, DNA marker DL2000; Fig1a, Lane 1 and 2, l-LS in pSB1C3 plasmids; Fig 1b, Lane1 and 2, d-LS in pSB1C3 plasmids; Plasmid are digested by EcoRI and PstI.

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	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	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.

Fig.2 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.

1. Judging standard of the single sample K-S test
   If P > 0.05, it has normality, otherwise it doesn’t have normality.
2. 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.

3. Output the results

Table.2 paring sample test

	Paring difference
				95 % confidence interval of difference
	Mean	Standard deviation	The standard error of mean	Lower limit	Upper limit
paired control - test	-46.31579	45.38484	10.41200	-68.19058	-24.44100

Table.3 paring sample test

	T statistics	Freedom	P value
Paired control-test d	-4.448	18	0.000

Bace 16

1. agarose gel electrophoresis of bace16

We did restriction endonuclease analysis to test if we had successfully synthesized bace16 with pBAD promoter(BBa_K206000) in its upstream region. We digested the plasmid with restriction enzymes EcoRI and PstI. Then we did agarose gel electrophoresis and found that the plasmid was digested into two fragments, one of which was about 1300bp long(Fig.3). This proves that we have successfully built the biobrick bace16-pSB1C3 as we planned earlier.

Fig.3 the agarose gel electrophoresis of gene bace16, the bands pointed by arrows represent bace16 genes. Lane M, DNA marker DL2000; Lane 1 and 2, bace16-pSB1C3 plasmids digested by EcoRI and PstI.

2. SDS-PAGE of Bace16

After we successfully built this biobrick, we transferred the vectors into E.coli BW25113 to express Bace16 protein. SDS-PAGE was done to testify the expression of rMpL protein. The expression of Bace16 was induced by L-Arabinose at the concentrations ranging from 8uM-12uM, culturing was at 26℃ in shaking incubator for over 5h. As the literature indicated[1], Bace16 is an extracellular secretion protein, so we gathered the supernatant to test the exist of it. According to the SDS-PAGE figure, we failed to expressed Bace16 in E.coli BW25113(Fig.4,5)

Fig 4. SDS-PAGE of Bace16 Lane 1, molecular weight standards (kDa); lane 2-5, ammonium sulfate precipitation of the supernatant after 0, 8 \(\mu\)M, 10uM and 12 \(\mu\)M L-Ara induction respectively; lane 6-9, total bacterial lysate after 0, 8 \(\mu\)M, 10 \(\mu\)M and 12 \(\mu\)M L-Ara induction respectively.

Fig.5 SDS-PAGE of Bace16 Lane 1, molecular weight standards (kDa); lane 2-5, the supernatant after 0, 8 \(\mu\)M, 10 \(\mu\)M and 12 \(\mu\)M L-Ara induction respectively, after 11000 rpm for 10 min; lane 6-9,the sediment.

3. Discussion

The molecular mass of Bace16 mature protein is 28kDa, but the SDS-PAGE analysis did not show the 28kDa band. There are mainly two reasons for this result.

First, as the Bace16 protein is an extracellular secretion protein in nature, we gathered the supernatant and did ammonium sulfate precipitation, if the ammonium sulfate precipitation was not successfully conducted, we would not observe the target band in our experiment.

Bace16 is a serine protease, it contains a presequence signal peptide of 30 amino acids and a propeptide of 77 amino acids^[2]. We add the presequence and prosequence when we designing the biobrick, thusmay lead the bace16 not be expressed well in E. Col. Besides, it might be degrased by the proteins from E. coli, so we couldn’t detect the expression of Bace16 in E. coli.

rMpL

1. Agarose gel electrophoresis of rMpL

First, we did restriction endonuclease analysis to test if we had successfully synthesized rMpl gene with pBAD promoter(BBa_K206000) in its upstream region. We digested the plasmid with restriction enzymes EcoRI and PstI. Then we did agarose gel electrophoresis(AGE) and found that the plasimd was digested into two fragments, one of which was about 700bp long, as we expected(Fig.6). It proves that we have successfully built the vector rMpL-pSB1C3 as we planned earlier.

Fig.6 The agarose gel electrophoresis of gene rMpL, the bands pointed by arrows represent rMpL genes.Lane M,DNA marker; Lane 1-4, rMpL-pSB1C3 plasmids digested by EcoRI and PstI. sites.

Besides, we have built another vector including rMpL gene. The backbone of this vector is also pSB1C3, but the promoter of rMpL has been changed into a constitutive promoter(BBa_J23100). We also did restriction endonuclease analysis on this vector either. The agarose gel electrophoresis figure shows that we successfully built this part too.

2. SDS-PAGE

After we successfully built the vectors, we transferred the vectors into different E.coli strains depending on the kinds of vectors .Vectors with pBAD promoter were transferred into E.coli BW25113 and vectors with constitutive promoter were transferred into E.coli BL21 to express rMpL protein respectively. After rMpl gene was expressed in the bacteria, we did SDS-PAGE to testify the expression of rMpL protein. And according to the SDS-PAGE figure, we expressed rMpL in both E.coli strains.(Fig.2,3)

Fig.7 SDS-PAGE of rMpL

Lane 1, molecular weight standards (kDa); lane 2-5, supernatant after 1.2, 1.2, 1.0, 1.0 \(\mu\)M L-Ara induction respectively; lane 7, supernatant after 0 \(\mu\)M L-Ara induction. Lane 8: supernatant of the pSB1C3; Lane 9,10,12,13,14: homogenate after 1.2, 1.2, 1.0, 1.0, \(\mu\)M L-Ara induction respectively; lane 15: homogenate of the pSB1C3.

Fig.8 SDS-PAGE of rMpL (constitutive promoter ) decoloration for five hours

Fig.9 SDS-PAGE of rMpL (constitutive promoter) decoloration for ten hours

3. Nematoxicity Test

At last, we did nematoxicity test to find if rMpL protein did be able to prevent C.elegans from developing to adulthood from larva. As we showed in the microscopic figures below, nematodes treated with recombinant bacteria show significant difficulty in developing to adulthood, such as the length of the nematodes and the motion activity of nematodes. While the control group showed the opposite reaction, the worms can develop to adulthood normally. And as for the motility of the nematodes, the nematodes which ate the bacteria-expressed rMpL moved slower than the control group. The following figures shows the differences between the experimental groups and the control groups.

Fig.10 After nematodes ate the bacteria only with pSB1C3

Fig.11 After nematodes ate the bacteria with rMpL

4. Discussion

During our experiment, we found an interesting phenomenon. When we changed our pBAD promoter to constitutive promoter, and growth of the E.coli became much slower.(Fig.12). pSB1C3-BW25113 is the bacteria with the pBAD promoter and the pSB1C3-BL21 is the bacteria with constitutive promoter(BBa_J23100). We can easily find the difference.

Fig.12 Cultivating for 5 hours

Fig.13 Cultivating for 10 hours

We think it may be because rMpL could do harm to the E.coli themselves. Hence,it verifies that the pBAD promoter is more suitable than a constitutive promoter.

Circuit design and test

1. pcyA+ho1

We connected gene pcyA（BBa_I15009）and ho1（BBa_I15008）together along with the constitutive promoter(BBa_J23100) through overlap PCR. After we transformed the restriction-ligation product into E.coli TOP10, we did colony PCR to test if we had successfully synthesized this biobrick. The agarose gel electrophoresis showes that this biobrick was successfully constructed(Fig.4). And this work was helped by BIT-CHINA, for more information, please see the collaboration section.

Fig.14 the agarose gel electrophoresis of pcyA+ho1, the bands pointed by arrows represent the expected result. Lane M, DNA marker DL2000; Lane 1 to 12, colony pcr of pcyA+ho1 biobrick; Lane 11, presents an approximately 1500bp of pcyA+ho1 band which is as expected.

2. PompC+RFP

In order to test whether our light regular system can work well, we added a reporter gene GFP downstream the promoter PompC (Fig5.), transformed this biobrick to TOP10 strain, and tested the basal activity of promoter PompC.

Fig15. The agarose gel electrophoresis of PompC+RFP and PompC+6N+gp35, the bands pointed by arrows represent the expected result. Lane 1,DNA marker DL5000;Lane 2, PompC+RFP; Lane3, PompC+6N+gp35.

When we just transformed this circuit into the E.coli Top 10, we found some of the colony became red. It indicated the colony had express RFP protein. Without the regulation of the OmpR, the promoter PompC started the transcription of the downstream target gene. And then we did a sequencing towards the colony which expressed the RFP. The result indicated the PompC-RBS-RFP circuit led to the expression of the RFP.

Fig.16 E.coli TOP10 transformed the PompC-RBS-RFP circuit

We detected the sensibility of red colony. We set RFP coding device(BBa_J04450）, RBS-rfp-terminator(BBa_K516032) and pSB1C3 as the control group. We plated 100 \(\mu\)L the overnight culture on the LB medium consisting chloramphenicol (34 \(\mu\)g/mL) and cultivated them in constant temperature foster box at 37℃. Half of them were under shading treatment. After 12 hours we observed the colony.

Fig.17 Detecting the sensibility to light (from left to right: PompC-rfp, pSB1C3, rfp and RBS-rfp-ter)

Fig.18 The results of the light sensibility experiment (4-1 from left to right: pSB1C3, RBS-rfp-ter, PompC-rfp and rfp 4-2 from left to right: PompC-rfp with light，PompC-rfp without light 4-3 from left to right: PompC-rfp without light, rfp with light)

We found that all the plates transformed PompC-rfp and rfp became red. But the PompC-rfp only expresses RFP faintly and the differences between light and dark are not obvious. It showed that the individual PompC-rfp biobrick was not sensitive to light. And it also indicated the Pompc promoter was at background level in E.coli TOP10.

The reason may be that the promoter PompC is upstream of the ompC porin gene in nature. The regulation of ompC is determined by the EnvZ-OmpR osmosensing machinery. EnvZ phosphorylates OmpR to OmpR-P. At high osmolarity, EnvZ is more active, creating more OmpR-P. OmpR-P then binds to the low-affinity OmpR operator sites upstream of ompC^[3].

The essence is that the EnvZ protein senses the mediun osmolarity and then forces the OmpR protein to take one of two alternative structures, which positively regulate OmpC synthesis^[4].

So we designed an experiment to detect, under the normal level of the Envz, the trend of E.coli PompC with the change of osmotic pressure.

Overnight cultures of Top10 strains transformed with PompR-rfp, rfp, pSB1C3 and RBS-rfp-Ter respectively grown at 37 °C in LB medium containing appropriated antibiotics were diluted at least 1:100 in the medium and incubated at 37 °C as fresh cultures. After their OD₅₉₀ reached 0.2~0.4, the fresh culture was diluted 1 : 3 into 4 mL of LBON medium (1g Tryptone, 1g Yeast Extract in 100mL H₂O). For osmolarity conditions, the cultures were diluted with NaCl supplemented medium to the final concentration of 0% ,0.25%, 0.50% and 1% (wt/vol). After 12 hours of induction, the results are as follows.

3. PompR+6N+gp35

We inserted the gp35 gene into the downstream region of the PompC promotor by means of 3A Assembly. After we transformed the restriction-ligation product into E.coli TOP10, we did colony pcr. The agarose gel electrophoresis showed that this biobrick was successfully constructed (Fig15.).

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2. Day R M, Thalhauser C J, Sudmeier J L, et al. Tautomerism, acid‐base equilibria, and H‐bonding of the six histidines in subtilisin BPN′ by NMR[J]. Protein Science, 2003, 12(4): 794-810.
3. Mizuno T M S. Isolation and characterization of deletion mutants of ompR and envZ, regulatory genes for expression of the outer membrane proteins OmpC and OmpF in Escherichia coli.[J]. Journal of Biochemistry, 1987, 101(2):387-396.
4. Hall, M.N. & Silhavy, T.J. (1981) J. Mol. Biol. 151, 1-15