Biosensor based on overexpressed D1 protein

Relazione del progetto "BEEP": Assembly and application of photosystem II-based biosensors for large scale environmental screening of specific herbicides and heavy metals (QLK3-CT-2001-01629) coordinatore Dr. Maria Teresa Giardi Overexpression of D1 Chlamydomonas reinhardtii in E. coli strains Wild type and mutagenised psbA genes were cloned from Chlamydomonas reinhardtii, which were previously provided by partner 2. The polymerase chain reaction (PCR) was performed on total DNA by proof reading Taq (Promega) to avoid any copy mistake. The forward primer container: a site-specific recombination stretch from ? phage (attB1), a Shine-Dalgarno signal and a sequence matching with the 5' coding region of psbA gene. The reverse primer spanned the psbA 3' region, comprising the stop codon and a trailer, followed by a terminal ? phage site-specific recombination sequence (attB2). The attB sequences are necessary to clone the gene in the correct orientation and efficiently in the Gateway technology (Invitrogen). All amplified sequences were checked for correct DNA copying and subsequently cloned into the Gateway expression vector driven by a salt inducible promoter. As a mandatory step, D1 protein overexpression of the intron-less wild type psbA was attempted. The gene expression was salt induced in recombinant E. coli strains which were sampled after 2,3,4 hrs from the induction. Total proteins were extracted from bacterial broth, bacterial lisates, cell inclusive bodies and their membranes. They were electrophoresed, blotted onto nitrocellulose membrane and hybridised to anti-rabbit D1 protein and revealed a chromatic reaction using by anti-rabbit phosphatase conjugated secondary antibody. The strongest signal was detected in all the fractions containing soap washed membranes of inclusive bodies. Isolation and characterization of psbA genes from distinct Medicago sativa ecotypes The DNA of three distinct ecotypes (T, K and R see materials and methods) was isolated and full length gene cloning was performed by PCR, using primers designed on alfalfa psbA gene (accession number: X04973). Proof reading Taq polymerase was used to carry out the experiments to avoid any copy mistake. DNA sequencing and alignment analyses indicated that 5 distinct point mutations distinguished K and R lines, however two of them were silent mutations (positions: 42 and 126). At position 145 an alanine residue appears in K line, while a valine residue is in R line. At positions 166 and 333 glycine residues are found in K, while glutamic acid in R line. PCR products from T lines is being currently sequenced and analysed. Materials and Methods Chlamydomonas reinhardtii strains and culture Strains containing the wild type and mutagenised psbA genes (IL, wild type; Epi1, Epi2, D170E, G178S, D170E/G178S and SK, mutants) were provided and maintained on solid and liquid media according to the provider (partner 2) Plant material and growth conditions Seeds of three alfalfa ecotypes were provided by the Bulgarian University...... a high-yielding line (T), a selected drought tolerant line (R) derived from water stressed T populations selection and the drought tolerant commercial variety Krasnovodopadskaya (K). Seeds were germinated and grown in peat under controlled conditions (continuous light with fluorescent lamps, Philips TLD 25,000 lux) as reported in Ivanova et al. J. Plant Physiol. 150: 224-227, 1997. Leaves were sampled and immediately frost into liquid nitrogen and stored at -80°C. Gene cloning and overexpression PCR experiments were performed on total DNA isolated from alfalfa using Plant DNAzol Kit (Invitrogen) according to the manufacturer's instructions. PCR primers were designed from functional conserved domains of psbA genes. Final PCR conditions were: 100 ng of DNA, 1?M of each primer, 0.5 mM dNTPs, Taq proof reading (Promega) 2.5 U, 1/10 of 10X Taq Buffer, 2.5 mM MgCl2, in a final volume of 50 ?l. Cycling conditions included an initial cycle of 95°C for 5 min followed by 35 cycles of 95°C for 1 min, 60°C for 30 sec and 72°C for 1 min, followed by a final extension cycle at 72°C for 5 min. The 1110 bp fragment was cloned into pGEM-T easy vectors, sequenced and aligned for polymorphisms analysis by ClustalW (European Bio-Informatics Service). Chloroplast DNA was isolated from algae as previously described (Newman et al., 1990. Genetics 126: 875-888). Details on PCR primers are in the results. PCR physical conditions were: 5 min at 94°C followed by 94°C for 30 sec, 50°C for 1 min, 90 sec at 68°C (35 cycles) and a final elongation of 5 min at 68°C using a Platinum Pfx Taq (Invitrogen). Amplified fragments were cloned into pDONR?201 by site-specific recombination according to the manufacturer's instructions . After kanamycin selection, psbA containing vectors were transferred in a pDEST14 vector which allows gene expression in ampicillin selected- recombinant BL21-SI E. coli. We produced E.coli lines overxpressing pbsA genes from psbA from Chlamidomonas IL (wild type), Epi1, Epi2, D170E, G178S, D170E/G178S ed SK mutants. Western analysis Strains harbouring the wild type psbA gene (derived from IL line) were induced by salt treatment following instruction and protein content analysed. Isolation of total protein and purification from distinct cell fractions and components (bodies of inclusion) were carried out according to Sambrook and Maniatis 1984. Protein content was quantified by standard procedures (BIORAD). 50?g of total proteins was loaded on gels containing a urea gradient, 12% Acrilamide, 0,4M Tris-HCl pH 8.8, 0,05% ammonium persulfate (APS), 5?l Temed for the running gel, and 0,05M TrisHCl pH 6.8, 0,04% APS, 3,1?l temed for stacking gel. Running conditions were: 200V for about 45 min. To check for equal loading, one control electrophoresis gel was stained with Comassie Blu, while another equal has been transferred to nitrocelloluse membrane and hybridised with the anti-D1 polyclonal antibody.