Toxin manufacture
Introduction
Biological p class="p1"esticides can be divided into two typ class="p1"es: small comp class="p1"ounds and biological macromolecules. On one hand, small comp class="p1"ounds are more p class="p1"rone to be absorbed by termites while more costly to p class="p1"roduce. On the other hand, macromolecules are easier and cheap class="p1"er to p class="p1"roduce 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 p class="p1"roteins. We p class="p1"lan to overexp class="p1"ress avermectin in its host Strep class="p1"tomyces avermitilis and exp class="p1"ress three kinds of toxic p class="p1"rotein 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, p class="p1"lease go to(CNC的主页面)
Avermectin manufacture
Judging that many toxic small comp class="p1"ounds are harmful to human being, we choose avermectin, which is highly sp class="p1"ecific to insect and does no harm to human. For one thing, being a secondary metabolite p class="p1"roduced by Strep class="p1"tomyces avermitilis, avermectin is regulated by an 80kb gene cluster(1), making it difficult to exp class="p1"ress in other standardized strains, for instance, Escherichia coli. For another, the avermectin yield in wild typ class="p1"e S. avermitilis strain is comp class="p1"aratively low(1). Nevertheless, we p class="p1"lan to engineer the wild S. avermitilis to imp class="p1"rove the yield of avermectin, embed the engineered strain with CNC and feed termites with CNC embedded S. avermitilis.
AVERMECTIN: EFFECTIVE AND BROAD-SPECTRUM PESTICIDE
For years, p class="p1"eop class="p1"le always adop class="p1"t the organochlorine p class="p1"esticides such as chlordane and mirex to achieve p class="p1"revention and control of termites, but these organochlorine p class="p1"esticides will p class="p1"roduce p class="p1"ollution and p class="p1"otential harm to the environment. Avermectin is a new typ class="p1"e of high efficient biological p class="p1"esticide, which has good control effect to the termites and other p class="p1"ests, and no p class="p1"ollution to the environment(1).
HOST OF AVERMECTIN——Strep class="p1"tomyces avermitilis
Strep class="p1"tomyces avermitilis, a soil-dwelling gram-p class="p1"ositive microorganism, is a rich source of numerous secondary metabolites. It’s a kind of Actinomycetes with staghorn-like hyp class="p1"ha (figure 2). Now it has been industrialized to p class="p1"roduce the commercially imp class="p1"ortant antip class="p1"arasitic agent avermectin(2). Early in 2003, the comp class="p1"lete genome of Strep class="p1"tomyces avermitilis had been sequenced(3).
In p class="p1"ast years, scientists had been trying to transform gene into S.avermitilis. Until 1989, gene transformation in S.avermitilis was achieved through conjugation(超链接到下面的conjugation) between E.coli strains(eg, s17-1)and S.avermitilis(4)¬. However, the efficiency was limited by the methyl-sp class="p1"ecific 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 (超链接到下面的ET12567介绍) Now conjugation and strain ET12567 has been ubiquitously adop class="p1"ted 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 p class="p1"aid to increase its yield, including develop class="p1"ing genome-minimized hosts, engineering the metabolic network(2), etc. In our p class="p1"roject, we p class="p1"lan to overexp class="p1"ress three genes, frr, orfX, metK in S. avermitilis to imp class="p1"rove the yield of avermectin.
CIRCUITS DESIGN
We have constructed three circuits to imp class="p1"rove the yield of avermectin(figure 3). PROMOTER: ermEp class="p1" We chose ermEp class="p1", a strong constitutive p class="p1"romoter, to overexp class="p1"ress the three genes in S.avermitilis. It should be noticed that ermEp class="p1" can only be exp class="p1"ressed in S.avermitilis strains instead of Escherichia coli or any other chassis.
BACKBONE: PL96 and PL97
` PL96 and PL97 are two high-cop class="p1"y vectors we used to overexp class="p1"ress our target genes. We get these vectors through commercial p class="p1"urchase. These vectors have p class="p1"UC18 and p class="p1"IJ101 rep class="p1"lication origins for high-cop class="p1"y p class="p1"lasmid number in Escherichia coli and S.avermitilis, resp class="p1"ectively, and the oriT (RK2) allows the efficient and convenient p class="p1"lasmid transfer from E.coli to S.avermitilis(6).
To be noticed, we use sp class="p1"ecial antibiotic ap class="p1"aramycin to choose final transformants. And there are ap class="p1"aramycin resistent gene acc in the backbone.
EXPRESSION:
In order to construct and exp class="p1"ress the three gene in S.avermitilis, we have adop class="p1"ted two hosts, E.coli DH5αand 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 p class="p1"lenty 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-sp class="p1"ecific restriction system in S.avermitilisi.(超链接到上面的介绍页面)
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 p class="p1"rovides a conjugative or mobilizable genetic element that is most often a p class="p1"lasmid or transp class="p1"oson(8). In laboratories, successful transfers have been rep class="p1"orted from bacteria to yeast(9), p class="p1"lants(10), mammalian cells(11), etc. In our p class="p1"roject, we use the conjugation between E.coli ET12567 and S.avermitilisi to overexp class="p1"ress three target genes.
To see the results of exp class="p1"ression and toxic exp class="p1"eriment on termites, p class="p1"lease go to (results页面)
CIRCUITS CONSTRUCTION
STEP ONE: PCR
We amp class="p1"lify the target gene from the genome of S.avermitilisi by PCR. The p class="p1"rimer and PCR p class="p1"rogram can be seen in our biobrick p class="p1"ages（超链接到biobrick）.
STEP TWO: TA CLONING
We use TA cloning to efficiently clone the PCR p class="p1"roducts. In TA cloning, we use p class="p1"MD19-T Vector, a vector transformed from p class="p1"UC19 vector, to imp class="p1"rove the efficiency of digestion and connection. As a result, we get three recombinant vectors of target genes and p class="p1"MD19-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, HindⅢ to digest the three recombinants and backbone PL97 and connect the corresp class="p1"onding p class="p1"roduct. Then we get the target p class="p1"lasmids.
For more detailed p class="p1"rotocols, p class="p1"lease go to(超链接到p class="p1"rotocol).
Toxic p class="p1"rotein manufacture
Cellulose has sp class="p1"ecific structure feature, based on which we successfully achieve self-assembly between CNCs and bacteria in aqueous solution driven by multivalent interactions esp class="p1"ecially the multip class="p1"le hydrogen bonds. This p class="p1"age will discuss the main theories and methods of CNC carriers.
Structure of cellulose
In order to kill the termites, we have chosen four typ class="p1"es of insecticidal toxic p class="p1"roteins, resp class="p1"ectively Tc p class="p1"rotein 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 corresp class="p1"onding vectors, successfully exp class="p1"ressed these p class="p1"roteins in Escherichia coli BL21(DE3) and fed the termites with the raw engineered BL21 and that embedded with CNC. For more information about CNC, p class="p1"lease go to(CNC的主页面)
HOST OF TOXIN -- Photorhabdus luminescens
Photorhabdus luminescens, one kind of gram-negative bacteria, is cap class="p1"able of p class="p1"roducing and releasing a variety of insecticidal and bactericidal toxins. Living in symbiosis with nematodes, “the bacteria are released and start to p class="p1"roduce 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 p class="p1"redicted to encode 4839 kinds of p class="p1"rotein(12). And many of them are toxic p class="p1"roteins, most of which remain functionally unclear. Although they are toxic to insects and many other bacteria, Photorhabdus luminescens belongs to Risk Group class="p1" 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 ap class="p1"p class="p1"lication of nematodes for controlling insect p class="p1"ests also showed that EN and their symbiotic bacteria (Photorhabdus luminescens) are safe to human” and “EN-based bio-p class="p1"esticides were exemp class="p1"ted from registration in many countries, including USA and all Europ class="p1"ean 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 comp class="p1"lex gene cluster, which makes it difficult to exp class="p1"ress in other standardized hosts, for instance Escherichia coli. Hence, on account of cost and safety, we chose four typ class="p1"es of single-gene regulated toxic p class="p1"rotein, tcdA1, tcdB1, Plu0840 and Plu1537, instead of small molecules because the former is easier to manip class="p1"ulate 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-p class="p1"ositive bacteria.
Tcs are comp class="p1"osed of TcA, TcB, and TcC. TcA is sup class="p1"p class="p1"osed to p class="p1"erforate the membrane by forming channel outside-in and translocating the toxic enzymes into the host. Meanwhile the TcB and TcC coop class="p1"erate with a syringe-like mechanism during membrane insertion(14).
In a 2008 study, researchers exp class="p1"ressed tcdA1 and tcdB1 in Enterobacter cloacae and fed the termites with E. cloacae to control termites(15). Insp class="p1"ired by their exp class="p1"eriment, we chose to exp class="p1"ress tcdA1 (Unip class="p1"rot: Q7N7Y9_PHOLL) and tcdB1(Unip class="p1"rot: Q7N7Z0_PHOLL) to kill termites. For more details, p class="p1"lease go to p class="p1"arts 的网页链接
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 p class="p1"rotein has 30% p class="p1"redicted amino acid sequence similarity to a 13.6 kDa insecticidal crystal p class="p1"rotein cry34Ab1(figure 12) in Bacillus thuringiensis (Unip class="p1"rot: Q939T0_BACTU), which belongs to Bt crystal p class="p1"rotein family, it may have similar toxic effect with cry34Ab1 Bt p class="p1"rotein.
Bt p class="p1"rotein may be the most well-known toxic p class="p1"rotein till now. It is widely used in transgene p class="p1"lants to kill the larvae of worm. It also “interacts with membranes to form p class="p1"ores”(17). And there are abundant evidences to ensure the safety of Bt p class="p1"rotein(更详细？).
We have successfully cloned the p class="p1"lu1537 gene and exp class="p1"ressed the Plu1537 toxin p class="p1"rotein in E.coli BL21(DE3), for more details, p class="p1"lease 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 p class="p1"lu0840 in the P. luminescens TT01 genome has 55% sequence identity with an enterotoxin Ast from Aeromonas hydrop class="p1"hila, therefore may p class="p1"lay a similar role. (see figure 13)
In 2001, researchers studied the function of enterotoxin Ast from Aeromonas hydrop class="p1"hila, concluded that it p class="p1"layed an imp class="p1"ortant role in A. hydrop class="p1"hila-induced gastroenteritis in a mouse model(18).
We have successfully cloned the p class="p1"lu0840 gene and exp class="p1"ressed he Plu0840 toxin p class="p1"rotein in E.coli BL21(DE3), for more details, p class="p1"lease go to
CIRCUITS DESIGN
As disp class="p1"layed in figure 14, we have constructed three devices to exp class="p1"ress corresp class="p1"onding toxic p class="p1"roteins, p class="p1"lu1537 (BBa_K1668010), p class="p1"lu0840 (BBa_K1668009) and tcdA1 (BBa_K1668008) (注：p class="p1"arts name超链接到p class="p1"arts页面
PROMOTER: p class="p1"Bad(BBa_I0500)
We chose arabinose inducible p class="p1"romoter p class="p1"Bad (BBa_I0500) because it’s not only of medium strength with arabinose up class="p1" to certain concentration, but also have little leakage. Moreover, the p class="p1"Bad p class="p1"romoter is rep class="p1"ressed by glucose, giving the exp class="p1"ression more controllability. In order to p class="p1"romote exp class="p1"ression, we chose one of the strongest RBS in Parts Registry (BBa_B0034).
BACOBONE: p class="p1"SB1C3
EXPRESSION:
We adop class="p1"ted tandem exp class="p1"ression of toxin and rep class="p1"orter mCherry (BBa_K1668011) to roughly judge whether toxin is exp class="p1"ressed.
We use E.coli DH5α to get p class="p1"lenty recombinants in high quality and quantity. Then we transform the p class="p1"ositive recombinants into E.coli BL21(DE3) for high-quality exp class="p1"ression.
To see the results of exp class="p1"ression and toxic exp class="p1"eriment on termites, p class="p1"lease go to (results页面)
CIRCUITS CONSTRUCTION
STEP ONE: PCR
We amp class="p1"lify the target gene from the genome of S.avermitilisi by PCR. We also clone the arabinose inducible p class="p1"romoter p class="p1"Bad from Part Registry. The p class="p1"rimer and PCR p class="p1"rogram can be seen in our biobrick p class="p1"ages（超链接到biobrick）.
STEP TWO: BACKBONE DIGESTION
We digest the p class="p1"art 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 class="p1" cloning kit of Vazyme comp class="p1"any to assemble the target gene and backbone. The mechanism is showed in figure 15.
For more detailes about scarless assembly and any other p class="p1"rotocols, p class="p1"lease go to(超链接到p class="p1"rotocol)
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