Team:ZJU-China/Design/Toxinmanufacture

Toxin Manufacture

Introduction

Biological pesticides can be divided into two types: small compounds and biological macromolecules. On one hand, small compounds are more prone to be absorbed by termites while more costly to produce. On the other hand, macromolecules are easier and cheaper to produce whereas sometimes not as effective as small molecules. Hence, to kill termites more efficiently and effectively, we choose both - insecticidal small molecule avermectin and several toxic proteins. We plan to overexpress avermectin in its host Streptomyces avermitilis and express three kinds of toxic protein in Escherichia coli BL21(DE3) . Then we embed the engineered S. avermitilis and E.coli with CNC carrier and fed termites with the CNC embedded bacteria. For more information about CNC, please go to the main page of CNC .

Avermectin manufacture

Judging that many toxic small compounds are harmful to human being, we choose avermectin, which is highly specific to insect and does no harm to human. For one thing, being a secondary metabolite produced by Streptomyces avermitilis , avermectin is regulated by an 80kb gene cluster(1), making it difficult to express in other standardized strains, for instance, Escherichia coli . For another, the avermectin yield in wild type S. avermitilis strain is comparatively low(1). Nevertheless, we plan to engineer the wild S. avermitilis to improve the yield of avermectin, embed the engineered strain with CNC and feed termites with CNC embedded S. avermitilis .

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AVERMECTIN: EFFECTIVE AND BROAD-SPECTRUM PESTICIDE

For years, people always adopt the organochlorine pesticides such as chlordane and mirex to achieve prevention and control of termites, but these organochlorine pesticides will produce pollution and potential harm to the environment. Avermectin is a new type of high efficient biological pesticide, which has good control effect to the termites and other pests, and no pollution to the environment(1).

Figure 1 abstract process of self-assembly

HOST OF AVERMECTIN - Streptomyces avermitilis

Streptomyces avermitilis , a soil-dwelling gram-positive microorganism, is a rich source of numerous secondary metabolites. It's a kind of Actinomycetes with staghorn-like hypha (figure 2). Now it has been industrialized to produce the commercially important antiparasitic agent avermectin(2). Early in 2003, the complete genome of Streptomyces avermitilis had been sequenced(3).

Figure 2 the picture of Streptomyces avermitilis under scanning electron microscope.

In past years, scientists had been trying to transform gene into S.avermitilis. Until 1989, gene transformation in S.avermitilis was achieved through conjugation between E.coli strains (eg, s17-1 )and S.avermitilis (4) ¬. However, the efficiency was limited by the methyl-specific restriction system in S.avermitilisi , which show strong restriction to gene methylated in normal E.coli strains (5). Eventually, high efficiency conjugation was achieved till the introduction of methylase-negative donor strain E.coli ET12567 Now conjugation and strain ET12567 has been ubiquitously adopted in the gene transformation of S.avermitilis.

PROBLEMS AND SOLUTIONS

Environmentally friendly though avermectin is, the yield of avermectin in wild S. avermitilis doesn't fulfill our needs. Many efforts have been paid to increase its yield, including developing genome-minimized hosts, engineering the metabolic network(2), etc. In our project, we plan to overexpress three genes, frr, orfX, metK in S. avermitilis to improve the yield of avermectin.

CIRCUITS DESIGN

We have constructed three circuits to improve the yield of avermectin(figure 3). PROMOTER: ermEp We chose ermEp, a strong constitutive promoter, to overexpress the three genes in S.avermitilis . It should be noticed that ermEp can only be expressed in S.avermitilis strains instead of Escherichia coli or any other chassis.

Figure 3 the circuits constructed for yield improvement of avermectin in S.avermitilis .

BACKBONE: PL96 and PL97

PL96 and PL97 are two high-copy vectors we used to overexpress our target genes. We get these vectors through commercial purchase. These vectors have pUC18 and pIJ101 replication origins for high-copy plasmid number in Escherichia coli and S.avermitilis , respectively, and the oriT (RK2) allows the efficient and convenient plasmid transfer from E.coli to S.avermitilis (6).

Figure 4 the map of plasmid backbone PL96.

Figure 5 the map of plasmid backbone PL97.

To be noticed, we use special antibiotic aparamycin to choose final transformants. And there are aparamycin resistent gene acc in the backbone.

EXPRESSION:

In order to construct and express the three gene in S.avermitilis , we have adopted two hosts, E.coli DH5α and E.coli ET12567 . Then the target vectors are transferred from E.coli ET12567 to S.avermitilis by conjugation.

PRIMARY HOST: E.coli DH5α

As usual, we use E.coli DH5α to get plenty of recombinants in high quality and quantity.

INTERMEDIA HOST: E.coli ET12567

, E.coli ET12567 is a methylase-negative donor strain first used by MacNeil in 1988(7). And we use E.coli ET12567 to demethylation the recombinants to better suit the methyl-specific restriction system in S.avermitilisi.

CONJUGATION:

Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. During conjugation the donor cell provides a conjugative or mobilizable genetic element that is most often a plasmid or transposon(8). In laboratories, successful transfers have been reported from bacteria to yeast(9), plants(10), mammalian cells(11), etc. In our project, we use the conjugation between E.coli ET12567 and S.avermitilisi to overexpress three target genes.

To see the results of expression and toxic experiment on termites, please go to results page .

CIRCUITS CONSTRUCTION

STEP ONE: PCR

We amplify the target gene from the genome of S.avermitilisi by PCR. The primer and PCR program can be seen in our biobrick pages .

STEP TWO: TA CLONING

We use TA cloning to efficiently clone the PCR products. In TA cloning, we use pMD19-T Vector, a vector transformed from pUC19 vector, to improve the efficiency of digestion and connection. As a result, we get three recombinant vectors of target genes and pMD19-T.

STEP THREE: DIGESTION AND CONNECTION

We digest the three recombinants and backbone PL96 with restriction enzymes NdeI, XbaI, then connect the fragments and backbone. Similarly, we use NdeI, Hind III to digest the three recombinants and backbone PL97 and connect the corresponding product. Then we get the target plasmids.

Figure 7 the sketch map of PL96 plasmid construction.

Figure 8 the sketch map of PL97 plasmid construction.

For more detailed protocols, please go to protocol .

Toxic protein manufacture

In order to kill the termites, we have chosen four types of insecticidal toxic proteins, respectively Tc protein tcdA1, tcdB1, bt-like Plu0840 and enterotoxin-like Plu1537, from Photorhabdus luminescens TT01, a bacterium of native toxin storehouse. Then we cloned these genes from the genome of TT01 , constructed corresponding vectors, successfully expressed these proteins in Escherichia coli BL21(DE3) and fed the termites with the raw engineered BL21 and that embedded with CNC. For more information about CNC, please go to the main page of CNC.

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HOST OF TOXIN -- Photorhabdus luminescens

Photorhabdus luminescens , one kind of gram-negative bacteria, is capable of producing and releasing a variety of insecticidal and bactericidal toxins. Living in symbiosis with nematodes, the bacteria are released and start to produce toxins that eventually kill the insect after insect larvae are invaded by nematodes, thereby generating a food resource for bacteria and nematodes (12).

The whole genome of strain TT01, which has been sequenced in 2003, is predicted to encode 4839 kinds of protein(12). And many of them are toxic proteins, most of which remain functionally unclear. Although they are toxic to insects and many other bacteria, Photorhabdus luminescens belongs to Risk Group 1 according to DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) and has no toxic effect on human being at all. More than 50 years of field application of nematodes for controlling insect pests also showed that EN and their symbiotic bacteria ( Photorhabdus luminescens ) are safe to human and EN-based bio-pesticides were exempted from registration in many countries, including USA and all European countries (13).

TOXIN PROTEIN IN P. luminescens TT01

Numerous toxins as there are in the genome of P. luminescens TT01 , many of them have never been studied. Moreover, many small-molecule toxins are regulated by complex gene cluster, which makes it difficult to express in other standardized hosts, for instance Escherichia coli. Hence, on account of cost and safety, we chose four types of single-gene regulated toxic protein, tcdA1, tcdB1, Plu0840 and Plu1537, instead of small molecules because the former is easier to manipulate and less risky to the environment.

tcdA1: PORE FORMING PROTEIN of Tc TOXIN FAMILY

The most remarkable toxin family till now is the Tc family, which are widely distributed among different gram-negative and gram-positive bacteria.

Tcs are composed of TcA, TcB, and TcC. TcA is supposed to perforate the membrane by forming channel outside-in and translocating the toxic enzymes into the host. Meanwhile the TcB and TcC cooperate with a syringe-like mechanism during membrane insertion(14).

In a 2008 study, researchers expressed tcdA1 and tcdB1 in Enterobacter cloacae and fed the termites with E. cloacae to control termites(15). Inspired by their experiment, we chose to express tcdA1 (Uniprot: Q7N7Y9_PHOLL) and tcdB1(Uniprot: Q7N7Z0_PHOLL) to kill termites. For more details, please go to parts

Plu1537: Bt HOMOLOGOUS TOXIC PROTEIN

The exact function of Plu1537 is still unclear, but a research in 2009 indicated that Plu1537 had insecticidal activity against Galleria larvae (16).

Judging that the Plu1537 protein has 30% predicted amino acid sequence similarity to a 13.6 kDa insecticidal crystal protein cry34Ab1(figure 12) in Bacillus thuringiensis (Uniprot: Q939T0_BACTU), which belongs to Bt crystal protein family, it may have similar toxic effect with cry34Ab1 Bt protein.

Bt protein may be the most well-known toxic protein till now. It is widely used in transgene plants to kill the larvae of worm. It also “interacts with membranes to form pores”(17). And there are abundant evidences to ensure the safety of Bt protein(更详细？).

We have successfully cloned the plu1537 gene and expressed the Plu1537 toxin protein in E.coli BL21(DE3) , for more details, please go to

Plu0840: ENTEROTOXIN Ast HOMOLOGOUS PROTEIN

The exact function of Plu0840 is also unclear. A 2007 study confirmed that Plu0840 had weak oral toxicity against two kinds of moth ( S. litura and S. exigua )(13).

Sequence analysis showed that the plu0840 in the P. luminescens TT01 genome has 55% sequence identity with an enterotoxin Ast from Aeromonas hydrophila, therefore may play a similar role. (see figure 13)

In 2001, researchers studied the function of enterotoxin Ast from Aeromonas hydrophila, concluded that it played an important role in A. hydrophila-induced gastroenteritis in a mouse model(18).

We have successfully cloned the plu0840 gene and expressed he Plu0840 toxin protein in E.coli BL21(DE3) , for more details, please go to the next page.

CIRCUITS DESIGN

As displayed in figure 14, we have constructed three devices to express corresponding toxic proteins, plu1537 (BBa_K1668010), plu0840 (BBa_K1668009) and tcdA1 (BBa_K1668008)

PROMOTER: pBad(BBa_I0500)

We chose arabinose inducible promoter pBad (BBa_I0500) because it's not only of medium strength with arabinose up to certain concentration, but also have little leakage. Moreover, the pBad promoter is repressed by glucose, giving the expression more controllability. In order to promote expression, we chose one of the strongest RBS in Parts Registry (BBa_B0034).

BACOBONE: pSB1C3

EXPRESSION:

We adopted tandem expression of toxin and reporter mCherry (BBa_K1668011) to roughly judge whether toxin is expressed.

We use E.coli DH5α to get plenty recombinants in high quality and quantity. Then we transform the positive recombinants into E.coli BL21(DE3) for high-quality expression.

To see the results of expression and toxic experiment on termites, please go to (results页面)

CIRCUITS CONSTRUCTION

STEP ONE: PCR

We amplify the target gene from the genome of S.avermitilisi by PCR. We also clone the arabinose inducible promoter pBad from Part Registry. The primer and PCR program can be seen in our biobrick pages .

STEP TWO: BACKBONE DIGESTION

We digest the part BBa_J06702, mCherry with RBS in front and double terminator behind, with restriction enzyme XbaI to make a linearized backbone.

STEP THREE: SCARLESS ASSEMBLY

We use the MultiS_one step cloning kit of Vazyme company to assemble the target gene and backbone. The mechanism is showed in figure 15.

For more detailes about scarless assembly and any other protocols, please go to protocol