Patent Application: US-58618509-A

Abstract:
this disclosure provides a method to reduce cell damage caused by near uv light absorption of algal or cyanobacterial cultures . the algal or cyanobacterial cells are transformed to express one or more fluorescent proteins , that absorb the harmful uv or near uv wavelengths and emits wavelengths that are photosynthetically more active . the photosynthetic pigments of the transgenic algal or cyanobacterial cell culture will then absorb the photosynthetically active light emitted by the fluorescent proteins . accordingly the harmful effects of the uv and near uv radiation are reduced and the photosynthetic activity of the algal or cyanobacterial cells is improved .

Description:
algae and cyanobacteria with biotechnological utility are chosen from among the following , non - exclusive list of organisms chlamydomonas reinhardtii , pavlova lutheri , isochrysis sp . cs - 177 , nannochloropsis oculata cs - 179 , nannochloropsis like cs - 246 , nannochloropsis salina cs - 190 , tetraselmis suecica , tetraselmis chuii and nannochloris sp . as representatives of all algae species . synechococcus pcc7002 , synechococcus wh - 7803 , thermosynechococcus elongatus bp - 1 as representatives of all cyanobacterial species . the algae come from a large taxonomical cross section of species ( table 1 ) many genes that in higher plants and chlorophyta are encoded in the nucleus are encoded on the chloroplast genome ( plastome ) in the chromobiota red lineage algae ( grzebyk , et al ., 2003 ) de novo synthesized blue fluorescent protein ( bfp )- azurite , a5cdna ( mena et al ., 2006 ) or other fluorescence proteins such as dsred , for enhancing algal and cyanobacterial photosynthesis and / or preventing cell damage caused by short wavelengths were cloned under the control of the hsp70 - rbcs2 promoter or other constitutive promoters and 3 ′ rbcs2 terminator for algae ( fig2 and 3 ). more than one fluorescent protein can be cloned in tandem to achieve stacking , leading to optimal utilization of the total light spectrum reaching the culture . genes encoding more than one fluorescent protein can be functionally stacked in a sequential manner , or by co - transformation . the methodologies used in the various steps of enabling the invention are described below : algae cells in 0 . 4 ml of growth medium containing 5 % peg mw6000 were transformed with , for example , 1 to 5 μg of the plasmid described in example 1 , by the glass bead vortex method ( kindle , 1990 ). the transformation mixture was then transferred to 10 ml of non - selective growth medium for recovery and incubated for at least 18 h at 25 ° c . in the light . cells were collected by centrifugation and plated at a density of 10 8 cells per 80 mm petri dish . transformants were grown on fresh tap or sgii agar plates containing a selective agent for 7 - 10 days at 25 ° c . fresh algal cultures are grown to mid exponential phase in artificial seawater ( asw )+ f / 2 media . cells are then harvested and washed twice with fresh media . after resuspending the cells in 1 / 50 of the original volume , protoplasts are prepared by adding an equal volume of 4 % hemicellulase ( sigma ) and 2 % driselase ( sigma ) in asw and are incubated at 37 ° c . for 4 hours . protoplast formation is tested by calcofluor white non - staining . protoplasts are washed twice with asw containing 0 . 6m d - mannitol ( sigma ) and 0 . 6m d - sorbitol ( sigma ) and resuspended in the same media , after which dna is added ( 10 μg linear dna for each 100 μl protoplasts ). protoplasts are transferred to cold electroporation cuvettes and incubated on ice for 7 minutes , then pulsed in an ecm830 electroporation apparatus ( btx , harvard apparatus , holliston , mass ., usa ). a variety of pulses is usually applied , ranging from 1000 to 1500 volts , 10 - 20 msec per pulse . each cuvette is pulsed 5 - 10 times . immediately after pulsing the cuvettes are placed on ice for 5 minutes and then the protoplasts are added to 250 μl of fresh growth media ( without selector ). after incubating the protoplasts for 24 hours in low light at 25 ° c . the cells are plated onto selective solid media and incubated under normal growth conditions until single colonies appear . a fresh algal culture is grown to mid exponential phase in asw + f / 2 media . a 10 ml sample of the culture is harvested , washed twice with dulbecco &# 39 ; s phosphate buffered saline ( dpbs , gibco , invitrogen , carslbad , calif ., usa ) and resuspended in 250 μl of buffer r ( supplied by digital bio , nanoentek inc ., seoul , korea , the producer of the microporation apparatus and kit ). after adding 8 μg linear dna to every 100 μl cells , the cells are pulsed . a variety of pulses is usually needed , depending on the type of cells , ranging from 700 to 1700 volts , 10 - 40 msec pulse length ; each sample is pulsed 1 - 5 times . immediately after pulsing , the cells are transferred to 200 μl fresh culture media ( without selector ). after incubating for 24 hours in low light at 25 ° c ., the cells are plated onto selective solid media and incubated under normal culture conditions until single colonies appear . a fresh algal culture is grown to mid exponential phase in asw + f / 2 media . 24 hours prior to bombardment cells are harvested , washed twice with fresh asw + f / 2 and resuspended in 1 / 10 of the original cell volume in asw + f / 2 . 0 . 5 ml of each cell suspension is spotted onto the center of a 55 mm petri dish containing 1 . 5 % agar solidified asw + f / 2 media . plates are left to dry under normal growth conditions . bombardment is carried out using a pds 1000 / he biolistic transformation system according to the manufacturer &# 39 ; s ( biorad laboratories inc ., hercules , calif ., usa ) instructions using m10 tungsten powder ( bioradlaboratories inc .) for cells larger than 2 microns in diameter , and tungsten powder comprised of particles smaller than 0 . 6 microns ( fw06 , canada fujian jinxin powder metallurgy co ., markham , on , canada ) for smaller cells . the tungsten is coated with linear dna . 1100 or 1350 psi rupture discs are used . all disposables ( unless otherwise noted ) are supplied by biorad laboratories inc . after transformation the cells are incubated under standard culture conditions for 24 hours , followed by transferring the cells onto selective solid media at a density of 10 4 cells per 90 mm diameter plates , and incubated under normal growth culture until single colonies appear . for transformation to synechococcus pcc7002 , cells are cultured in 100 ml of bg - 11 30 turks island salts liquid medium ( http :// www . crbip . pasteur . fr / fiches / fichemedium . jsp ? id = 548 ) at 28 ° c . under white fluorescent light and subcultured at mid exponential growth . to 1 . 0 ml of cell suspension containing 2 × 10 8 cells , 0 . 5 - 1 . 0 μg of donor dna ( in 10 mm tris / 1 mm edta , ph8 . 0 ) is added , and the mixture is incubated in the dark at 26 ° c . overnight . after incubation for a further 6 h in the light , the transformants are selected on bg - 11 + turks island salts agar plates containing a selection agent until single colonies appear . for quantification of the transgene expression products , proteins are isolated from the algal cells utilizing a buffer containing 750 mm tris ph 8 . 0 , 15 % sucrose ( wt / vol ), 100 μm β - mercaptoethanol and 1 mm phenylmethylsulfonylfluoride ( pmsf ). samples are then centrifuged for 20 min at 13 , 000 × g at 4 ° c ., with the resulting supernatant used in western immunoblotting . western immunoblotting is carried out as described by cohen et al . ( 1998 ) using a rabbit anti - rcfp polyclonal pan antibody that detects any of the entire panel of gfp - like reef coral fluorescent proteins ( clontech , palo alto , calif ., usa ) and an alkaline phosphatase - labeled goat anti - rabbit secondary antibody ( sigma ). proteins for in vitro bfp assays are prepared in the same fashion except that the crude lysate is centrifuged for 30 min at 40 , 000 × g at 4 ° c . to remove contaminating thylakoids . microtiter assays are carried out on volumes of 100 μl with samples diluted in protein extraction buffer . protein concentrations are determined using bio - rad protein assay reagent ( bio - rad laboratories inc ). for screening for transgenes expressing high levels of bfp mrna , total rna is isolated using either qiagens &# 39 ; s plant rneasy kit ( qiagen , hilden , germany ) or the trizol reagent ( invitrogen , carlsbad , calif ., usa ). cdna is synthesized using 3 μg total rna as a template with an oligo - dt primer for algae and a specific 3 ′ primer for cyanobacteria , and superscript ™ ii reverse transcriptase ( invitrogen , carlsbad , calif ., usa ) according to the manufacturer &# 39 ; s instructions . presence of bfp - azurite dna was tested by pcr using bfp - azurite specific primers ( sequence in example 1 ). redtaq dna polymerase ( sigma ) was used for the pcr amplification . a 1 kb dna ladder was used as dna size marker ( fermentas , md ., usa ). fluorescent proteins transform high energy , damaging ( near - uv ) wavelengths into lower energy , longer , less damaging ( blue to red ) wavelengths . fluorescent proteins with overlapping excitation and emission spectra , can convert light from any wavelengths ( near - uv , green ) poorly used by photosynthetic pigments into photosynthetically more active wavelengths . in order to test the hypothesis that cells expressing synthetic genes encoding fluorescent proteins will be more efficient using whole light spectra reaching the culture , cells expressing the bfp - azurite or any other type of fluorescent protein are compared to wild type cells . to assess the contribution of fluorescent proteins to cell photosynthetic efficiency , cells are illuminated with narrow band light with a peak at excitation wavelength of the fluorescent proteins . ( e . g . a near - uv led — light emitting diode ) emitting at 375 ± 5 nm ( fig4 ). photosynthetic activities of the transgenic algae are examined and compared to those of the wild types by measuring oxygen evolution in the light and oxygen consumption in the dark , using clark type electrodes ( pasco scientific , roseville , calif ., usa ). a setup for comparative evaluation of oxygen evolution was built , allowing simultaneous measurements of 8 algal samples illuminated at different intensities and wavelengths . temperature is maintained using a water - bath with circulator ( model cb 8 - 30e , heto lab instruments ). cells of eukaryotic marine cultures ( e . g . isochrysis galbana , phaeodactylum tricornutum and nannochloropsis sp .) and transformants thereof are cultured on artificial seawater ( asw ) medium ( wyman et al ., 1985 ) supplemented with f / 2 ( guillard and ryther , 1962 ). marine cultures are grown at 22 - 25 ° c . with a 16 / 8 h light / dark period . fresh water cultures ( e . g . chlamydomonas reinhardtii ) and transformants thereof are cultured photoautotrophically on in liquid medium , using mineral medium as previously described ( harris , 1989 ), supplemented with 5 mm nahco 3 , with continuous shaking and illumination at 22 ° c . cells of marine cyanobacteria ( e . g . synechococcus pcc 7002 ) and transformants thereof are cultured in medium bg - 11 + turks island salts liquid medium ( http :// www . crbip . pasteur . fr / fiches / fichemedium . jsp ? id = 548 ). cyanobacteria are cultured at 25 ° c . under continuous white light , with constant co 2 - air bubbling . in order to test the hypothesis that cells expressing synthetic genes encoding fluorescent proteins are more efficient than the wild type capable of using sunlight , we compare algae expressing fluorescent proteins to wild type cells in ambient sunlight . for example , the growth rate of wild type and bfp - azurite transformants cultured in par ( photosynthetically active radiation — i . e . 450 - 750 nm light ) and par + near - uv is measured using direct cell counts . culture density is measured daily for a period of ten days . the growth rate of wild type and dsred transformants cultured in sunlight is measured using direct cell counts . culture density is measured daily for a period of ten days . algae and cyanobacteria expressing fluorescent proteins have increased photosynthetic activity and growth rate compared to the wild type at the tested wider light spectrum containing near - uv . the invention is now described by means of various non - limiting examples : the bfp - azurite sequence ( mena et al ., 2006 ) was artificially synthesized using the published sequence ( seq id no : 1 ) with modifications according to the codon usage of p . tricornutum ( bfp - pt ) ( seq id no : 2 ) and the green algae c . reinhardtii ( bfp - cr ) ( seq id no : 3 ) and with the addition of bstbi and bamhi restriction sites at its ends . the gene was cloned into pgem - t vector ( promega , madison , usa ) and then ligated into the bstbi / bamhi restriction sites of psi103 ( sizova et al ., 2001 ) replacing the aphviii selectable marker gene , generating the plasmid psi - bfp . in this plasmid the bfp - azurite gene is under the control of the hsp70a / rbcs2 promoter and 3 ′ rbcs2 terminator . parental strain c . reinhardtii cc - 425 was co - transformed with the psi - bfp - pt plasmid and linearized plasmid pjd67 , containing the structural gene ( arg7 ) of the argininosuccinate lyase to complement the arg2 locus ( davies et al . 1994 , 1996 ). c . reinhardtii colonies were selected on tap medium without arginine . approximately 35 colonies that grew without arginine were transferred to liquid tap medium and screened for psi - bfp construct using pcr with primers ( fig5 ): rna was extracted from positive colonies containing the psi - bfp construct for bfp expression monitoring by rt - pcr on cdna using the primers bfp - forward and bfp - reverse ( fig6 ). colonies expressing the bfp transcript are then screened for bfp expression as described in example 5 . in addition , the psi - bfp - pt / cr plasmid together with psi - pds plasmid containing the pds gene ( conferring resistance to the phytoene desaturase - inhibiting herbicide flurochloridone ) ( seq id no : 6 ) are co - transformed to nannochloropsis oculata cs - 179 and isochrysis sp . cs - 177 using the transformation methods described above . generation of synechococcus pcc7002 expressing the bfp - azurite gene under the control of the cyanobacterial rbcls promoter the bfp - azurite sequence ( mena et al ., 2006 ) is artificially synthesized to enhance stability using the published sequence ( seq id no : 1 ), but with modifications according to the preferred codon usage of synechococcus pcc7002 ( seq id no : 7 ) and with the addition of bamhi restriction sites at both ends . the gene is cloned into pgem - t vector ( promega , madison , usa ) and then transferred into the bamhi site of pcb4 plasmid ( deng and coleman , 1999 ) downstream to the synechococcus pcc 7002 rbcls promoter ( seq id no : 8 ) and upstream to rbcls terminator . likewise , similar constructs , made based on codon usage of other cyanobacterial species are generated and transformed into these species . the dsred gene is artificially synthesized using the published sequence ( accession number bae53441 ; seq id no : 9 ) with modifications according to the codon usage of the green algae c . reinhardtii ( seq id no : 10 ) and with the addition of bstbi and bamhi restriction sites at its ends . the gene is cloned into pgem - t vector ( promega , madison , usa ) and then ligated into the bstbi / bamhi restriction sites of psi103 ( sizova et al ., 2001 ) replacing the aphviii selectable marker gene , generating the plasmid psi - dsred . in this plasmid the dsred gene is under the control of the hsp70a / rbcs2 promoter ( seq id no : 11 ) and 3 ′ rbcs2 terminator . the gene product fluoresces green light to red wavelengths . the psi - dsred plasmid is co - transformed with psi103 containing the paromomycin resistance gene to c . reinhardtii cw15 ( cc - 400 ) and with psi - pds plasmid containing the pds gene ( conferring resistance to the phytoene desaturase - inhibiting herbicide flurochloridone ) to marine algae using the transformation methods described above . generation of synechococcus pcc7002 expressing the dsred gene under the control of the cyanobacterial rbcls promoter the dsred gene is artificially synthesized using the published sequence ( accession number bae53441 ; seq id no : 9 ) with modifications according to the codon usage of synechococcus pcc7002 ( seq id no : 12 ) and with the addition of bamhi restriction sites at both ends . the gene is cloned into pgem - t vector ( promega , madison , usa ) and then transferred into the bamhi site of pcb4 plasmid ( deng and coleman , 1999 ) downstream to the synechococcus pcc 7002 rbcls promoter ( seq id no : 8 ) and upstream to rbcls terminator . likewise , similar constructs , based on codon usage of other cyanobacterial species are generated and transformed into these species . bfp - azurite transformants are grown on fresh agar plates for 7 days at 25 ° c . colonies are transferred at equal concentrations to 200 μl culture media ( as described in the “ culture conditions ” section ) in 96 - well micro - well plates , and cultured under the conditions described in the “ culture conditions ” section , until they reach a substantial cell concentration (˜ 10 6 bfp fluorescence is excited at ˜ 380 nm and monitored at the emission of 450 nm . dsred and other fluorescent proteins are monitored according to their specific excitation and emission spectra . cells from cultures producing the highest fluorescent signal are collected and cultured as single cell colonies under 380 nm near - uv light ( duration and intensity are set at ld99 % of wild type cells ). surviving cells are then transferred for future culturing and further examination . screening for transformants expressing high level of bfp , dsred or other fluorescent proteins using western immunoblotting proteins from transformed algae and cyanobacteria cells with detectable levels of blue or other fluorescence are isolated from algae / cyanobacteria cells utilizing a buffer containing 750 mm tris ph 8 . 0 , 15 % sucrose ( wt / vol ), 100 μm β - mercaptoethanol and 1 mm phenylmethylsulfonylfluoride ( pmsf ). samples are then centrifuged for 20 min at 13 , 000 × g at 4 ° c ., with the resulting supernatant used for western immunoblotting . western immunoblotting is carried out as described by cohen et al . ( 1998 ) using an anti - rcfp polyclonal pan antibody primary antibody ( clontech , palo alto , calif ., usa ) and an alkaline phosphatase - labeled goat anti - rabbit secondary antibody ( sigma ). this polyclonal antibody recognizes the gfp - like family of proteins . one of the major goals in the field of production of photosynthetically generated materials ( such as oils , proteins , pigments and pharmaceuticals and other co - products ) is to utilize the whole spectrum of light reaching the photosynthetic cell , thus increasing photosynthetic efficiency and decreasing heating . in order to demonstrate that cells expressing synthetic genes encoding fluorescent proteins are more efficient using whole light spectra ( par and near - uv , or full sunlight ) reaching the culture , we compare photosynthetic efficiency of transformed algae or cyanobacteria expressing the bfp - azurite and / or any other single or multiple fluorescent proteins set to their respective wild type cultures . to assess the contribution of fluorescent proteins to cell photosynthetic efficiency , cells are illuminated with a narrow band light with a peak at excitation wavelength of the specific fluorescent protein . photosynthetic activity of the transgenic algae is examined and compared to that of wild type cells . oxygen evolution in the light and oxygen consumption in the dark is measured using clark type electrodes ( pasco scientific , roseville , calif ., usa ). algae and cyanobacteria expressing bfp - azurite have increased photosynthetic activity as measured by oxygen evolution . significant differences between oxygen evolution of algae and cyanobacteria expressing bfp - azurite and that of their respective wild type are observed when cells are illuminated with light at the excitation wavelength of bfp . in order to test that cells expressing synthetic genes encoding fluorescent proteins are more efficient at outdoor light conditions namely , ambient sunlight we compare growth rates of cultures expressing the bfp - azurite to that of wild type cells . growth rate at ambient conditions is determined by measuring culture density daily for a period of ten days . algae and cyanobacteria expressing bfp - azurite have increased photosynthetic activity and growth rate when compared to the wild type . chow k , tung w ( 1999 ) electrotransformation of chlorella vulgaris . plant cell reports 18 : 778 - 780 cohen a , yohn c b , bruick r k , mayfield s p ( 1998 ) translational regulation of chloroplast gene expression in chlamydomonas reinhardtii . methods enzymol 297 : 192 - 208 davies j p , yildiz f h , grossman a r ( 1994 ) mutants of chlamydomonas with aberrant responses to sulfur deprivation . plant cell 6 : 53 - 63 davies j p , yildiz f h , grossman a r ( 1996 ) sacl , a putative regulator that is critical for survival of chlamydomonas reinhardtii during sulfur deprivation . embo 15 : 2150 - 2159 . deng m d , coleman j r ( 1999 ) ethanol synthesis by genetic engineering in cyanobacteria . appl environ microbiol 65 : 523 - 528 gilmore a m , larkum a w , salih a , itoh s , shibata y , bena c , yamasaki h , papina m , van woesik r ( 2003 ) simultaneous time resolution of the emission spectra of fluorescent proteins and zooxanthellar chlorophyll in reef - building corals . photochem photobiol 77 : 515 - 523 grzebyk , d ., o . schofield , p ., falkowski , and j . bernhard ( 2003 ) the mesozoic radiation of eukaryotic algae : the portable plastid hypothesis . j . phycol . 39 : 259 - 267 ) guillard , r . r . and ryther , j . h . 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