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Applications of BBa_K417000

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UNIQ70018ea962557676-partinfo-00000000-QINU Group: ZJU-China 2015
Author: ZJU-China 2015
Preface:
The part BBa_K417000 just briefly introduced metK and its output SAMsynth. The specific function and principle of SAMsynth (S-adenosylmethionine synthetase) isn't mentioned. Furthermore, the 3D structure and other detailed information isn't included, which may bring much inconvenience to potential users. Thus, connected with our project, we added plenty of related information of metK and sincerely wish to give a hand to future users.
Summary:
1. characterize the output of metK in a novel chassis: Streptomyces avermitilis
2. create a visualization of S-adenosylmethionine synthetase
3. add the 2D PAGE maps for the output of metK in E.coli

Documentation

Part 1 characterization: the output of metK in Streptomyces avermitilis

Background

metK: S-adenosylmethionine synthetase encoding gene

metK is the gene encoding S-adenosylmethionine synthetase, which has been found in almost every organism. Its output catalyzes the formation of S-adenosylmethionine from methionine and ATP.

In Streptomyces avermitilis, it was found to stimulate the production of avermectins. When wild-type S. avermitilis strain ATCC31267 was transformed with pYJ02 and pYJ03, two metK expression plasmids, avermectin production was increased about 2.0-fold and 5.5-fold compared with that in the control strains, respectively.

As for the principle of improving the productivity, instead of changing cell growth or copy effect, metK stimulates the avermectin production by increasing the intracellular concentration of S-adenosylmethionine (SAM), an important intermediate product in avermectin production. However, there may be a maximum concentration of SAM for the production of avermectin in S. avermitilis, which means that SAM has no effect when its concentration achieve maximum.

The results of experiments in research paper showed that different metK expression levels have different influence on avermectin production in various S. avermitilis strains. The gene expression levels of metK in two engineered strain, GB-165 and 76-05, were much higher than those in wild-type strain, whereas the avermectin productivity in these two strains have not been significantly improved. It is probably because the high expression level of metK in engineered strains limited the improvement of avermectin productivity by overexpression of metK.

Fig.1 The picture of Streptomyces avermitilis under scanning electron microscope (SEM). Copyright all reserved ZJU-China 2015

host of avermectin——Streptomyces avermitilis

Streptomyces avermitilis, a soil-dwelling gram-positive microorganism, is a rich source of numerous secondary metabolites. Now it has been industrialized to produce the commercially important antiparasitic agent avermectin. Early in 2003, the complete genome of Streptomyces avermitilis had been sequenced.

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. 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. 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.

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.

Experiment: Avermectin manufacture in S. avermitilis

Purpose

We attempt to test the function of metK gene by improving the yield of avermectin in S. avermitilis. Because as it has been described in research paper, metK was found to stimulate the production of avermectins. For one thing, being a secondary metabolite produced by Streptomyces avermitilis, avermectin is regulated by an 80kb gene cluster, 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. Therefore, we plan to engineer the wild S. avermitilis with metK to improve the yield of avermectin.

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

Circuit design

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

backbone: PL96 and PL97

Fig.4 The plasmid map for PL96.

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Fig.5 The plasmid map for 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.

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 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 plentiful 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. And we use E.coli ET12567 to demethylation the recombinants to better suit the methyl-specific restriction system in S.avermitilis.

Fig.6 The sketch map of conjugation between E.coli ET12567 and 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. In laboratories, successful transfers have been reported from bacteria to yeast, plants, mammalian cells, etc.

In our project, we use the conjugation between E.coli ET12567 and S.avermitilisi to overexpress three target genes.

circuit construction

STEP ONE: PCR
We amplify the target gene from the genome of S.avermitilisi by PCR.

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 LIGATION

Fig.7 digestion and ligation in PL96.

Fig.8 digestion and ligation in PL97.

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 corresponding product. Then we get the target plasmids.

For more detailed protocols, please go to【超链接到队伍wiki的protocol页面】.

Result

Fig.9 Gel electrophoretic analyses of PCR products (A) and double enzyme digestion products (B and C). (A) 5-μl samples of the PCR products for metK, (B and C) 5-μl samples of the double enzyme digestion products were loaded onto a 1% BioRad Ready Agarose Mini Gel, then subjected to AGE. See (protocol) for AGE parameters. (B and C) Sizes of the NdeI and XbaI–cleaved assemblies were determined by AGE analysis. The DNA size standards was the DL2,000 DNA Marker (M1; TaKaRa, Cat#3427A) and 1kb DNA Ladder (Dye Plus) (M2; TaKaRa, Cat#3426A). Bands were visualized with a Shanghai Peiqing JS-380A Fluorescence Imager.

Gel electrophoretic analysis

We successfully constructed the plasmid (PL96 and PL97) containing metK (sequence is shown in the part BBa_K1668001) gene in E.coli DH5α and E.coli ET12567. Then by transforming it into S.avermitilis through conjugation, metK can be successfully expressed in a brand new chassis.

Part 2 a visualization of S-adenosylmethionine synthetase in E.coli

We have found the 3D molecular graphic (Fig.10) and interaction figure (Fig.11) of S-adenosylmethionine synthetase in NCBI database. http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=52023

Fig.10 3D molecular graphic of S-adenosylmethionine synthetase (MMDB ID: 52023)

Fig.11 the interaction figure of S-adenosylmethionine synthetase (MMDB ID: 52023 ). In the figure, ○ represents protein and ◇ represents chemical. The rhombus marked 1,2 and 3 represents phosphate ion, Co and K+, respectively.

Part 3 2DPAGE maps for the output of metK in E.coli

Fig.12 2DPAGE maps for S-adenosylmethionine synthetase in E.coli (SWISS-2DPAGE: P0A817)

We have found the 2DPAGE maps for S-adenosylmethionine synthetase in E.coli. You can click on a highlighted spot in the figure from the website listed below to access all the associated protein entries of the spot. http://world-2dpage.expasy.org/swiss-2dpage/viewer&map=ECOLI4.5-5.5&ac=P0A817

Uploads
http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=52023
http://world-2dpage.expasy.org/swiss-2dpage/viewer&map=ECOLI4.5-5.5&ac=P0A817

Reference: https://static.igem.org/mediawiki/parts/0/03/Overexpression_of_metK_shows_different_effects_on_avermectin_production_in_various_Streptomyces_avermitilis_strains.pdf

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Part:BBa_K417000:Experience