Molecular Characterization of Mosquitocidal Toxin (Surface Layer Protein, SLP) from Bacillus

Appl Microbiol Biotechnol

Molecular characterization of mosquitocidal toxin (Surface layer protein, SLP) from Bacillus cereus VCRC B-540.

Mani Chinnasamy1. Selvakumari Jeyaperumal1. Han YeonSoo2.

Jo YongHun2.Thirugnanasambantham Krishnaraj1.Sundarapandian Somaiah3. Poopathi Subbiah1,4

1Vector Control Research Centre (Indian Council of Medical Research), Department of Health Research, Ministry of Health and Family Welfare, Indira Nagar, Puducherry-605 006, India.

2Division of Plant Biotechnology, Institute of Environmentally-Friendly Agriculture (IEFA), College of Agriculture and Life Science, Chonnam National University, Gwangju, Republic of Korea

3 Department of Ecology and Environmental Sciences, School of Life-Sciences, Pondicherry University, Puducherry-605014, India.

4 Scientist-F/Deputy-Director (Senior Grade) & Corresponding author

Telephone No. +91-413-2272475, 2272397 & 2272948; Fax: 91-413-2272041;

E-mail:

Supplementary files

Fig S1. Vector map of pET-28a+

Fig S2. Multiple alignment of surface layer protein with closely related sequence Fig S3. SLP encoding gene from B. cereus cloned in E. coli DH5α

Fig S4. Hypothetical Illustration of Cloning of SLP into Expression system

Fig S5. SLP protein expression in host E. coli BL21(DE3)

Fig S6. Toxicity of purified SLP from B. cereus against Aedes albopictus (Larva source: Chonnam National University, S. Korea) Fig S7. Percentage of amino acid present in Surface layer protein from B. cereus VCRCB540

Table S1. Primers used in the present study

Table S2. Surface layer proteins used for phylogenetic tree

Table S3. Percentage identity and distance with surface layer protein from closely related existing strains

Fig S1. Vector map of pET-28a+

Fig S1: Vector map of pET-28a+ showed various restriction site and Nco I and Xho I restriction site was used for cloning and expression

Fig S2. Multiple alignment of surface layer protein with closely related sequence

Fig: The Slp (surface layer protein) encoding amino acid sequence was aligned and used for multiple sequence alignment with closely regaled sequence encoding amino acid of other bacterial species by using expasy (

Fig S3. SLP encoding gene from B. cereus cloned in E. coli DH5α

A. Surface layer protein amplification

B. Surface layer protein digestion with both Nco I and Xho I

C. Purification of Surface layer

protein

M- DNA Marker; 1-SLPM- DNA Marker; 1-Vector; 2- SLPM- DNA Marker; 1-SLP

A. Amplification of surface layer protein using gene specific primer; B. Digestion of amplified product with Nco I and Xho I; C. Purification of digested product

Fig S4. Hypothetical Illustration of Cloning of SLP into Expression system

This hypothetical illustration showed that the cloning and expression of SLP encoding gene into Expression system; T-Blunt (pGEM®-T)was used for cloning which (approximately 3015 bp) and also considered easy vector; whereas for expression, Vector pET-28a(+) (approximately 5369 bp) was used. NcoI and XhoI was used as restriction site.

Fig S5. SLP protein expression in host E. coli BL21(DE3)

A. Lane M: Protein marker; lane N.C (Negative control): Un-Insert E. coli BL21 (DE3); Lane P.C (Positive control): Inserted E. coli BL21; Lane: 2h, 4h, 6h, & 12h: different hours protein sample (2, 4, 6, & 12 hours respectively of 0.5 mM of IPTG; Lane: 2h, 4h, & 6h: different hours protein sample (2, 4 & 6 hours respectively of 1 mM of IPTG

B. Lane M: Protein marker;Lane P.C (Positive

control): Inserted E. coli BL21 (DE3); Lane 1-5:

Binding fraction; Lane 6-10: Washing fraction; Lane

11-17: Elution fraction.

Fig S6. Toxicity of purified SLP from B. cereus against Aedes albopictus (Larva source: Chonnam National University, S. Korea)

A.ControlPelletSupernatant

Moratilty

Fig. S6: A. The concentration of SLP-recombinant protein from supernatant of E.coli DE3 used against Aedes albopictus is 10µg/20ml water. Twenty five larvae per plate was used with three replicates. Larval mortality was recorded after 24 hours.

B. The bar diagram indicate the percentage mortality of larvae.

Fig S7. Percentage of amino acid present in Surface layer protein from B. cereus VCRCB540

* Newly isolated B. cereus VCRC B540 from marine fish gut content

Table S3. Percentage identity and distance with surface layer protein from closely related existing strains

Identity