Team:BNU-CHINA/Description

Introduction

Nematode has been up to over 5000 of 200 genus^[1]. Among them, over 100 species of nematodes are damaging the agricultural, forestal and economic crops of China who widely parasitize the roots, stems, leaves, flowers, buds and seeds of manifold plants, harming the development of agriculture and forestry seriously.

A newest research by USA indicates that the plant-parasitic nematodes damage leads to about 8 billion dollars’ losses to their croppers, which takes 12% of the whole value of the crop output. Meanwhile, according to incomplete statistics, the losses caused by paratrophy nematodes can reach 100 billion every year worldwide^[2]. China’s researchers have done some relevant surveys as well, and according to incomplete statistics, 17 provinces such as Anhui, Hainan, Hubei, Gansu, Zhejiang and Fujian have reported root knot nematode disease once, among which the morbidity of some severe regions in Shandong province can up to 2/3^[3]. Therefore we can note that plant-parasitic nematodes have brought out severe lost to global agriculture and forestry already.

Fig.1 Bursaphelenchus xylophilus

The size of plant-parasitic nematodes is usually too small for naked eyes to catch sight of. In addition, the present survey about the agricultural damage of nematodes are so limited that people often fail to notice and prevent it in time. The plant-parasitic nematodes are furnished with scalpellus functioning to stab into the plant cells to obtain nutrients after seeking out the host with the amphids on their forehead, which as a result, could damage the host as well as bring in pathogenic fungus and therefore causing complex harm to them. Up to now, the pathogenesis of plant-parasitic nematodes can be concluded in theory as follow:

1. The nematodes give raise to severe mechanical injury to the host plants when feeding on them with the scalpellus.
2. Other pathogen accompanied with the nematodes induce plant disease.
3. Some nematode secretion is toxic to the plants which as a consequence damages them.

In most canses, the mechanical damage by nematodes to host is a drop in the bucket, therefore the latter two theories are relatively common^[4].

The majority of harmful nematodes in agriculture and forestry belong to Tylenchida while a little belongs to Dorylaimida. Meloisogyne spp. Heterodera spp. Aphelenchoides composticola, Ditylenchus dipsaci are nematodes that gravely damage plants around the world. Among them, Meloisogyne spp. mainly destroy the roots of plants by forming root-knot to hinder their development, which in turns, causes the roots rot. According to statistics, a slight Meloisogyne spp. disease can lead to a 20%~30% output reduction, and 50%~70% even a total loss if it’s severe. Heterodera spp. also target the roots of plants primarily, weakening and decomposing the roots. Ditylenchus dipsaci damage the underground part such as the tuber, tuberous root, bulb of the plants which results in their rot and malformation while some local part over ground would also be influenced to turn to malformation, ,Aphelenchoides composticola such as Bursaphelenchus xylophilus and Aphelenchoides besseyi generally aim at the overground part of plants who infect the leaves and causing lesion and leaf tip drying, and more than that, infect the trunks to kill the whole plant rapidly.

Hence one can see that plant-parasitic nematode cause serious losses to a variety of agricultural crops worldwide. Since the traditional methods based on the use of nematocides and antihelminthic drugs are associated with major environmental and health concerns, the development of biocontrol agents to control nematodes is of major importance^[5]. In this case, our project is designed to control the losses caused by nematodes effectively and timely with biocontrol agents. And after the bait-kill system successfully established, we are looking forward to further expansion to other agricultural pests for the sake of achieving our ultimate goal – establishing a database containing attractant base and toxic protein base in allusion to all sorts of agricultural pests.

As to our project this year, we designed two models separately to attract and poison nematodes. We made E.coli synthesize limonene to bait plant-parasitic nematodes and then kill them with two kinds of toxic proteins Bace-16 and Mpl afterwards. Furthermore, we introduced a photo-regulated bi-direction transcription system to the whole system, by which means we would be able to regulate the .expression of attractant and toxic proteins through the control of light. That’s to say, the E.coli would express attractant to bait nematodes when exposed to light whereas express toxic proteins to kill them as a result of the promoter’s reverse in the dark.

Module 1: Bait

Limonene is a monoterpenoid, which is a raw material widely used in medicine and chemicals. There are two conformations in the nature. One is D configuration, the other is L configuration. Limonene can attract root knot nematodes of citrus or other plant parasitic nematodes^[6]. Our design based on the following two limonene synthase sequences which are named Citrus unshiu CitMTSE1 for d-limonene synthase and Mentha spicata 4S-limonene synthase. At the same time, we also designed another synthase sequence which can synthesize the isoprene GPP, the precusor substance of synthesizing the limonene. We transformed these two parts into E.coli to realize the expression of limonene in prokaryotic cell.

Module 2：Killer

Bace16 and MpL, these two protein have a good toxicity toward the nematodes. Bace16 is a kind of serine proteases from a bacterial parasite of the nematode named Bacillus sp. B16. After the expression of Bace16 has completed, the protein can be secreted out of the cell. This proteases could decompose the intestine protein of the nematodes thereby the nematodes will be killed^[7]. MpL is a novel lectin isolated from parasol mushroom. It is a kind of intracellular expression protein. MPL could bind with glycan of the nematodes specifically. So it can stop the growth of the nematodes from L1 phase to adults^[8].

Module3：Suicide

The system of attracting and killing above would carry out a series of functions. But at the same time, our recombination E.coli could suicide after a certain time. The suicide system is controlled by a regulatory switch. With the continual proliferation of the cells, the thallus can accumulate an organic named AHL. AHL can move across the cytomembrane freely. When it accumulates to a certain degree, it will combine with the intracellular proteins to induce the activation of the promoter of the suicide gene. Later the suicide system starts and the E.coli will die.

In order to attract and kill the nematodes, we also built a bidirectional photoinduced system. In this system, the expression of the attractive organics and the toxin are under different conditions. We have already known the promoter PompR is regulated by red light. When there is no light, the promoter combines with the ompR protein to function. We added the int gene behind the promoter, so the promoter would control the expression of the int protein. Under the effect of the int protein, the reversed promoter can reverse to activate the expression of the other side. In a word, we linked the genes of attractive organics and the toxin on each side of the Pcon respectively. And we controlled the different module by light.

Modeling

Our modeling can be divided into four parts. Firstly, in order to enable our project to be applied in life, we designed a device placed in soil, which can attract and kill nematodes by modified engineering bacteria inside. Secondly, assuming there is a farmland, we took advantage of nematodes’ movement analogue simulation to find the best position where the device should be placed. Thirdly, according to this concentration, we obtained the best size of the device by calculating. At last, we established a database to enlarge applied range of our method to kill other pests. We would appreciate that new synthetic biological and environment-friendly methods can be shared and improved with the science researchers all over the world.

Human Practice

During our project, we constantly did the communication and practice. We improved our plan by doing the human practice. At the same time, we also helped people understand the meaning of the synthetic biology and showed them the charm of the synthetic biology through our project. What’s more, we did a survey to publicise the damage of the plant parasitic nematodes and told the details to the farmers about the solutions. We interviewed the related experts, dropped in the countryside, analyzed the soil sample, communicated with other teams, did assistance and so on. The goal we would like to achieve is to help more people with the development of ourselves.

Improvement

Our Project

This year, our project uses the light regulation system to control the synthesis of bait and toxic protein. Our photoinduced system consists of the following four parts. They are Cph8（BBa_K592000），pcyA（BBa_I15009），ho1（BBa_I15008）and promoter PompC（BBa_R0082）.

1. cph8

The red light sensor (Cph8) is a fusion protein which is consisted of a phytochrome Cph1 and a histidine kinase domain, Envz-OmpR. Cph1 is the first member of the plant photoreceptor family that has been identified in bacteria. EnvZ-OmpR, a dimeric osmosensor, is a multidomain transmembrane protein. Cph1 can be inactivated under red light, Upon changes of extracellular osmolarity, EnvZ specifically phosphorylates its cognate response regulator OmpR, which, in turn, regulates the PompC. Cph8 can serve as a photoreceptor that regulates gene expression through PompC. Without red light, Cph1 is activated and it enables EnvZ-OmpR to autophosphorylate which in turn activates PompC. Under the exposure of red light, however, Cph1 is deactivated, inhibiting the autophosphorylation, thus turning off gene expression.

We insert RBS(B0035) and constitutive promoter(J23100) in front of the Cph8 sequences(K592000) to make sure that Cph8 can be expressed inside the E.coli.

2. pcyA+ho1

Moreover, Cph8 has to form chromophore with PCB biosynthetic genes (BBa_I15008 and BBa_I15009) in order to work, where the formation of PCB requires the gene pcyA and hol to function as accounted below. Hol is a sort of Iron red pigment oxidase which can oxidize the heme group using a ferredoxin cofactor, generating biliverdin IXalpha. And then, PcyA, a kind of ferredoxin oxidase from Synechocystis Sauv, functions to turn biliverdin IXalpha (BV) into PCB.

We connected gene pcyA（BBa_I15009）and ho1（BBa_I15008）together along with the constitutive promoter(BBa_J23100) to ensure a continuous work of the photoinduction system in our E.coli.

1.3 PompC

PompR is a OmpR-controlled promoter which can be positively regulated by phosphorylated OmpR. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription.

We insert PompR(BBa_R0082) sequences into the upstream region of our target gene int through restriction-ligation method, which together is connected to vector PSB1C3 afterwards, and therefore we are able to regulate the expression of in through the control of light.

Our Improvement Plan

According to the research, Cph8 is sensitive to red light, especially the wave length of red light is at 650nm. In order to improve the light sensitive characteristic of the Cph8 protein to satisfy the different requirements of this parts, such as regulating the efficiency of the promoter by control the wave length of the light, we plan to design an experiment to explore the sensibility of the Cph8 to wave length of 650nm nearby. Through this way we can control the output of the downstream product.

There are 20 experimental groups. We use the bandpass filter to control the wave length of the light from 550nm to 750nm. We set up every experimental group at a gradient of 20nm.

1. Materials

bandpass filter (550 /570…650 nm bandpass filters) , light (82 V, 300 W Philips FocusLine quartz bulb)

2. E. coli growth, light exposure and harvesting protocol

Figure 1

The protocol is performed over two consecutive days.

1. Late in the day, start a 37°C, shaking overnight culture from a −80 °C stock in a tube containing 3 mL LB medium and the appropriate antibiotics (50 μg/mL kanamycin, 50 μg/mL ampicillin and 34 μg/mL chloramphenicol).

2. After the overnight culture has grown for 10–12 h, prepare 100 mL LB medium. Add appropriate antibiotics to medium. Shake/stir the container to ensure the antibiotics are mixed well in the medium.

3. Measure the OD600 of the overnight culture.

4. Dilute the overnight culture 1mL into the 100mL LB + antibiotics. Shake/stir the container to ensure the cells are mixed well in the medium.

5. Place triangle bottles in the shaker and grow at 37°C with shaking at 250 rpm for 8 h. Use the narrow bandpass filter to set the wavelength of each bottles respectively. The intensity of light was measured in power units of watts per square meter using a EPP2000 UVN-SR calibrated spectroradiometer.

6. After 8 h of growth, harvest all test bottles by immediately transferring them into an ice-water bath. Wait 10 min for the cultures to equilibrate to the cold temperature and for gene expression to stop.

7. Approximately 1.5 h before stopping the experimental cultures, begin preparing a solution of phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH to 7.4). Prepare at least 1 mL for each culture to be measured via flow cytometry. At this time, begin preparing a 37°C water bath.

8. Filter the dissolved solution of PBS through a 0.22-μm 20-mL syringe filter.

9. Transfer 1 mL of the filtered PBS into one 5-mL cytometer tube per culture sample, and chill tubes in a rack in an ice-water bath.

10. Incubate the rack(s) of PBS + culture tubes in a 37 °C water bath for 1 h. Our measurements data is for the expression of RFP.

11. Transfer the rack(s) back into ice-water bath.

12. Wait 15 min, and then begin measuring each tube on a flow cytometer.

3. Expected Result

Figure 2

According to the existing literatures, what we expected is that the amount of the protein produced by experimental groups will increased at the early time, and then decreased later with the increasing of the light wave efficiency. The highest point is at around 650nm. According to the results, we can draw a line graph to describe the sensibility towards different wave length of Cph8. The following graph shows the tendency.

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Our Findings

We constructed the PompC-RBS-RFP circuit first (see parts page), when we just transform this circuit into the E.coli Top 10, we wondrously found some of the colony became red. It indicated the colony had express RFP protein. It means 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 the expression of the RFP.

Figure 3. 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 100uL the overnight culture on the LB medium consisting chloramphenicol (34ug/mL) and cultivated them in constant temperature foster box at 37℃. And half of them were under shading treatment. After 12 hours we observed the colony.

Figure 4. Detecting the sensibility to light (from left to right: PompC-rfp, pSB1C3, rfp and RBS-rfp-ter)

Figure 5. 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, all the plates transformed PompC-rfp and rfp became red. But the PompC-rfp only expresses RFP faintly and the differences with light or not 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.

Because in nature, this promoter PompC is upstream of the ompC porin gene. 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.

With increasing of the osmotic pressure, the expression of the rfp didn’t increase in experimental groups. So under natural condition, the expression of EnvZ-OmpR is too low to regulate the activity of PompC promoter. And then, from the pictures we can see the colony of experimental groups still became red. It shows that the existing of EnvZ-OmpR makes the PompC promoter become a little bit active under the natural conditions, the background level of the PompC is correspondingly higher. So if we want to try to control the expression of the downstream target gene of the PompC by using EnvZ-OmpR-PompC circuit, we’d better knock out the EnvZ-OmpR gene in the engineering bacteria first.

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