Engineering artificial photosynthetic life-forms through endosymbiosis
Table of Contents
Synechococcus elongatus strains were derived from S. elongatus PCC 7942 (Syn7942). This strain was obtained from Prof. Susan Golden’s lab (University of California San Diego, UCSD). We used S. cerevisiae ρ + NB97 (MATa leu2-3,112 lys2 ura3-52 his3ΔHindIII arg8Δ::URA3 [cox2-60]) as a host for S. elongatus endosymbionts. The S. cerevisiae-cox2-60 strain was obtained from the Schultz lab (Scripps Research).
S. elongatus cells were grown in sterile Erlenmeyer flasks containing liquid BG-11 medium. These cultures were shaken aerobically at 37 °C and 250 rpm under 3000 lux. Yeast cells were shaken aerobically at 30 °C and 250 rpm in YPD medium (1% yeast extract, 2% peptone, 2% glucose) containing 50 mg/L carbenicillin.
List of all the fusion selection media:
Selection medium I: 1% yeast extract, 2 % peptone, 3 % glycerol, 0.1 % glucose, 1 M sorbitol, 2 % agar, and 1X BG-11 salts.
Selection medium II: 1% yeast extract, 2 % peptone, 3 % glycerol, 0.1 % glucose, 1 M sorbitol, 2 % agar, 1X BG-11 salts, and 50 mg/ml carbenicillin.
Selection medium III: 1% yeast extract, 2 % peptone, 3 % glycerol, 1 M sorbitol, 2 % agar, 1X BG-11 salts, and 50 mg/ml carbenicillin.
NSII-recombinant S. elongatus mutant cultures were supplemented with 7.5 mg/L chloramphenicol. Unless noted otherwise, kanamycin-resistant S. elongatus mutant cultures were supplemented with 50 mg/L kanamycin and 2 mg/L L-methionine.
When noted, light-starved Syn7942 cells were inoculated (OD730 = 0.2) in BG-11 medium, wrapped in aluminum foil and incubated for 4 d, without shaking, at 30 °C. When noted, yeast media was supplemented with adenine sulfate (Alfa Aesar A16964-09, 20 mg/L), Antimycin A (Sigma A8674-25MG), or DCMU (A2B Chem AG00409). Yeast cells transformed with pML64 were selected on synthetic defined (SD) medium containing 0.67% yeast nitrogen base without amino acids (Sigma Y0626-250G), L-lysine (Sigma L5501-5G, 60 mg/L), L-histidine (Sigma H8000-10G, 20 mg/L), L-arginine (Sigma A-3704, 20 mg/L), uracil (Sigma U1128-25G) and 3% glycerol.
Construction of plasmids
Double-stranded and single-stranded DNA oligonucleotide fragments were purchased from Integrated DNA Technologies (IDT). Defined DNA fragments were amplified using PCR (Q5 Hot Start High-Fidelity 2X Master mix, NEB catalog # M0494S) and inserted into defined sites in the host vectors using Gibson assembly59. Double-stranded oligonucleotide sequences (gblocks) used for Gibson assembly are listed in Supplementary Data 1. Genomic DNA fragments used in cloning are listed in Supplementary Table 1. Cyanovectors were obtained from Prof. Susan Golden’s lab (UCSD). Where noted, coding sequences were codon-optimized for S. elongatus expression using IDT codon optimization software (https://www.idtdna.com/CodonOpt). All vectors were transformed into One Shot® ccdB Survival™ 2 T1R Chemically Competent Cells (Invitrogen A10460) according to manufacturer’s specifications. The oligonucleotides used in plasmid construction are listed in Supplementary Data 2. The plasmids pML3-pML28 are derived from the Cyanovector pCV0055, pML58 and pML62 are derived from the CyanoVector pCV0049. Vector maps are included in Supplementary Fig. 1, and detailed vector map links are provided in Supplementary Table 3.
pML3: pCV005518 was linearized by PCR using the oligonucleotides AM1216/AM1217. A gBlock of the UWE25-ntt1 gene codon optimized for S. elongatus was amplified by PCR using the oligonucleotides AM1214/AM1215. The amplified DNA fragment was inserted into linearized pCV0055 by Gibson assembly to afford pML3.
pML14: pML3 was linearized by PCR using the oligonucleotides AM1195/AM1312. A gBlock of the C. trachomatis incA gene codon-optimized for S. elongatus (Ctr-incA) was amplified by PCR using the oligonucleotides AM1313/AM1314 and inserted into linearized pML3 by Gibson assembly to afford pML14.
pML17: pML14 was linearized by PCR using the oligonucleotides AM1312/AM1351. A gBlock of the CT_813_CDO-1 gene was amplified by PCR using the oligonucleotides AM1352/AM1353 and inserted into linearized pML14 by Gibson assembly to afford pML17.
pML28: pML17 was linearized by PCR using the oligonucleotides AM1506/AM1507. A variant of the C. caviae incA gene codon-optimized for S. elongatus (Cca-incA) was amplified by PCR using the oligonucleotides AM1502/AM1503, purified and amplified once more using the oligonucleotides AM1504/AM1505. The amplicon was inserted into linearized pML17 by Gibson assembly to afford pML28.
pML62: pCV0049 was linearized by PCR using the oligonucleotides JC205/JC206. A 484-bp fragment of the 5′-end of the Syn7942 metA gene (Synpcc7942_0370) was amplified from purified Syn7942 genomic DNA using the oligonucleotides JC204/JC207. The amplicon was inserted into linearized pCV0049 by Gibson assembly and the construct was subsequently linearized using the oligonucleotides JC200/JC203. A 530-bp fragment of the 3′-end of Syncpcc7942_0370 was amplified from genomic DNA using the oligonucleotides JC201/JC202 and the amplicon was inserted into the linearized vector by Gibson assembly to afford pML62.
pML58: pML62 was linearized by PCR using the oligonucleotides AM1531/AM1532. The PCR product was purified and treated with T4 Polynucleotide Kinase (NEB #M0201S), T4 Ligase (NEB #MO202S) and DpnI (NEB #R0176S) for 1 h at room temperature to produce the ligated plasmid. The plasmid was then linearized by PCR using the oligonucleotides JC151/JC152. An origin of transfer (oriT) domain was amplified by PCR from the commercial plasmid pSET152 using the oligonucleotides JC153/JC154. The amplicon was inserted into the linearized plasmid by Gibson assembly to afford pML58.
pML63 is derived from the commercial plasmid pRS425, which was donated by the van der Donk lab (UIUC). The 2×Su9-MTS-LP-COX2-W56R expression cassette was first reported by Supekova and Schultz35.
pML59: pML14 was linearized by PCR using the oligonucleotides JC221/JC233. The linear PCR product was purified, amplified with JC221/JC226 and ligated by KLD reaction to afford the FLAG-tagged Ctr-incA construct pML59.
pML60: pML17 was linearized by PCR using the oligonucleotides AM1312/JC235 and ligated by KLD reaction. The construct was then linearized by PCR using the oligonucleotides JC223/JC233. The linear PCR product was purified, amplified with JC223/JC226 and ligated by KLD reaction to afford the FLAG-tagged CT_813 construct pML60.
pML63: pRS425 was linearized by PCR using the oligonucleotides JC249/JC248. The S. cerevisiae ACT1 promoter was amplified from purified S. cerevisiae genomic DNA by PCR using the oligonucleotides JC246/JC247. The amplified DNA fragment was inserted into linearized pRS425 by Gibson assembly and the construct was subsequently amplified by PCR using the oligonucleotides JC250/JC251. A gBlock of 2×Su9-MTS-LP-COX2-W56R was amplified by PCR using the oligonucleotides JC254/JC255 and the amplified DNA fragment was inserted into the linearized plasmid by Gibson assembly to afford pML63.
pML64: pML63 was amplified by PCR using the oligonucleotides JC128/JC273. The S. cerevisae TPI1 promoter was amplified from purified S. cerevisiae genomic DNA by PCR using the oligonucleotides JC272/JC127. The amplified DNA fragment was inserted into linearized pML63 by Gibson assembly to afford pML64.
Site-directed mutagenesis in cyanobacteria
Chromosomal integration of genes in Syn7942 was achieved using a modification of the method of Golden60. Briefly, a portion of Syn7942 overnight culture (15 mL) was centrifuged for 10 min at 3000 × g and 24 °C. The pellet was washed once with 10 mL NaCl (10 mM) and then resuspended in 0.3 mL BG-11 at room temperature. To this suspension was added purified plasmid (1.5 μL) containing genes flanked by NSII recombination sites. The mixture was added to a 1.5 mL microcentrifuge tube and shaken in the dark (12–16 h at 70 rpm). Cells transformed with NSII plasmids were added to glass test tubes containing liquid BG-11 (5 mL, room temperature) and antibiotic and cultured under normal conditions for 4 d, at which point surviving cells were added to Erlenmeyer flasks and grown under normal conditions. Cells transformed with pML58 were not viable when rescued in liquid BG-11 medium; instead, the cell-plasmid mixture was spread on BG-11-agar medium supplemented with kanamycin and L-methionine and incubated at 30 °C under 3000 lux for ≤10 days. The quality of S. elongatus cultures was evaluated regularly by microscopy, streaking the cells onto BG-11-agar and by PCR analysis of recombinant loci (oligonucleotides used for PCR analysis are listed in Supplementary Table 4).
SynJEC0 was generated by transformation of wild-type Syn7942 with pCV0055 to give a chloramphenicol-resistant recombinant mutant.
SynJEC1 was generated by transformation of wild-type Syn7942 with pML3 to give a recombinant mutant which ectopically expresses an ADP/ATP translocase from the NSII locus.
SynJEC2 was generated by transformation of wild-type Syn7942 with pML14 to give a recombinant mutant which ectopically expresses Ctr-incA and an ADP/ATP translocase from the NSII locus.
SynJEC3 was generated by transformation of wild-type Syn7942 with pML17 to give a recombinant mutant which ectopically expresses Ctr-incA, CT813 and an ADP/ATP translocase from the NSII locus.
SynJEC4 was generated by transformation of wild-type Syn7942 with pML28 to give a recombinant mutant which ectopically expresses Ctr-incA, CT813, Cca-incA and an ADP/ATP translocase from the NSII locus.
SynJEC5 was generated by transformation of SynJEC3 cells with pML58. The cells were plated on BG-11-agar medium supplemented with chloramphenicol (5 mg/L), kanamycin (5 mg/L) and L-methionine (2 mg/L), with individual colonies appearing within 10 days. Colonies were extracted and spotted on fresh BG-11 agar supplemented with the same components. After 7 days, the resulting dark green spots were added to liquid BG-11 (1 mL) and incubated under normal culturing conditions for Syn7942. The cells were passaged every 4 days into fresh BG-11 medium (5 mL) with increasing concentrations of kanamycin (5, 25 and finally 50 mg/L) in order to eliminate all chromosomal copies with intact Synpcc7942_0370. During each passage, the Synpcc7942_0370 locus was amplified by PCR using the oligonucleotides LL56/LL57. SynJEC5 cultures were considered ‘pure’ after 5 rounds of passaging, at which point only recombinant amplicons were produced by PCR. Methionine auxotrophy of SynJEC5 was evaluated by washing the cells in BG-11 medium and inoculating cultures containing chloramphenicol and kanamycin in the presence and absence of L-methionine (Supplementary Fig. 5a).
SynJEC9 was generated in a similar manner as SynJEC5 by transformation of SynJEC1 cells with pML58 instead of pML62. As with SynJEC5, the cells were plated on BG-11-agar medium supplemented with chloramphenicol (5 mg/L), kanamycin (5 mg/L) and L-methionine (2 mg/L). Individual colonies appeared within five days, which were then extracted and spotted on fresh BG-11 agar supplemented with kanamycin (5 mg/L) and L-methionine (2 mg/L). Afterwards, the cells were passaged through multiple rounds of growth in liquid BG-11 medium in the same manner as SynJEC5 until PCR analysis of genomic DNA with the oligonucleotides LL56/LL57 produced only a recombinant band at the Syncpcc7942_0370 locus (Supplementary Fig. 5a).
Measurement of ATP release by cyanobacteria mutants expressing ATP/ADP translocase after ADP challenge
In order to eliminate contaminant ATP, ADP solution (Sigma A2754) was treated with hexokinase according to the following protocol61. ADP (80 mM, pH 7.5) was incubated with D-glucose (200 mM), MgCl2 (2 mM) and hexokinase (Sigma H4502-500UN) (0.04 U/μL) at room temperature for 2 h. The solution was then filtered through an Amicon Ultra 0.5 column (14,000 × g, 15 min) to eliminate hexokinase.
SynJEC0 and SynJEC1 cells were grown for 3 d to reach densities of ~30,000,000 cells/mL. For each assay, 300,000,000 cells were harvested by centrifugation (3000 × g, 5 min, room temperature), the supernatant was aspirated and the pellet was washed once with 20 mM Tris-HCl. The cells were then incubated with ATP solution (Sigma G8877) (10 mM, pH 7.5) for 30 min at 37 °C, washed three times with 20 mM Tris-HCl to eliminate extracellular ATP. ADP (80 µM final concentration) was added and the cells were incubated statically at 37 °C. The mixtures were then centrifuged (10,000 × g, 5 min) and the supernatant ATP concentration was determined by luciferase assay (ATP determination kit, Life Technologies – #A22066). ATP standards (provided with the kit) were used obtain a calibration curve.
Introduction of mutant cyanobacteria to S. cerevisiae cells
We adapted a method for generating yeast-E. coli chimeras to be used with S. elongatus13,14. SynJEC0-9 mutants were grown under constant light, with shaking, for 4 d. After this time, the cells (30 mL) were harvested (3000 × g, 10 min, 24 °C), washed twice with BG-11 and resuspended in BG-11 (500 µL). S. cerevisiae cox2-60 cells were grown aerobically in YPD medium (120 mL) for 24 h. The yeast was harvested (4696 × g, 10 min, 24 °C), washed twice with sterile water, twice more with SCEM (1 M sorbitol, 13 mM β-mercaptoethanol) and resuspended in ice-cold sterile-filtered SCEM solution (10 mL) containing Zymolyase 100 T (15 mg per gram of yeast pellet). The suspension was incubated for 1 h at 37 °C to give spheroplasts. The suspension was then cooled on ice for 30 min and centrifuged for 10 min at 1500 × g and 4 °C. The pellet was washed twice, gently, with chilled SCEM and resuspended in SCEM (2.5 mL) The suspension would remain usable for fusions for at least 24 h if kept refrigerated.
The spheroplast suspension (750 µL) was mixed with chilled TSC buffer (10 mM Tris-HCl, 10 mM CaCl2, 1 M sorbitol, pH 8) (750 µL) and incubated for 10 min at 30 °C. This mixture was then centrifuged (1500 × g, 10 min) and the supernatant was carefully discarded. The spheroplasts were resuspended in room-temperature TSC buffer (120 µL) and sorbitol (4 M, 60 µL). Dense S. elongatus cell suspension (120 µL) was added quickly to the spheroplast suspension, mixed by tube inversion and incubated for 30 min at 30 °C. This mixture was then decanted into a round-bottom polypropylene tube containing PEG buffer (20% PEG 8000, 10 mM Tris-HCl, 2.5 mM MgCl2, 10 mM CaCl2, pH 8) (2 mL) and incubated without shaking at 30 °C for 45 min. The cells were centrifuged (1500 × g, 10 min, 24 °C), the supernatant was discarded and YPDS (YPD with 1 M sorbitol added) was added gently without disrupting the pellet. The cells were incubated under light without shaking for 2 h at 30 °C. After this time, the pellet was partially dislodged by flicking the tube. The mixture continued to incubate for 3 h with shaking (70 rpm), after which time the cells were harvested (1500 × g, 10 min, 24 °C), resuspended in 1 M sorbitol (300 μL)and plated on Selection-I medium. After drying for 5 min, a second layer of Selection-I medium was overlaid on top of the cells. The plates were incubated at 30 °C in a 12 h light-dark cycle for at least four days, until colonies appeared between the agar layers. The colonies were extracted from the agar, suspended in 1 M sorbitol and spotted on Selection-II medium. For subsequent rounds of propagation, cells were scraped from the surface of the agar, resuspended in 1 M sorbitol and spotted on Selection-III medium.
Cell count of S. cerevisiae cox2-60/cyanobacteria chimeras
Spots on Selection-III medium were removed manually from the agar plate and placed intact inside a 1.5-mL microcentrifuge tube. Sorbitol (1 M, 200 μL) was added to the surface of the agar and cells were removed by pipetting. The tube was briefly centrifuged (5 s) to remove the cell-containing flowthrough from the agar. Cells were then mounted on a reusable glass slide and counted in triplicate from a brightfield image using the Countess II FL Automated Cell Counter (Fisher cat. # AMQAF1000) per the manufacturer’s instructions.
Total genomic DNA isolation and PCR analysis
Total DNA isolation of chimeras was achieved using the Yeast DNA Extraction Kit (Thermo Fisher 78870) using the manufacturer’s protocol. Cells were scraped from the surface of the agar, harvested (5000 × g, 5 min), resuspended and incubated (65 °C, 10 min) with Y-PER Reagent (8 μL/mg pellet). The mixture was centrifuged (13,000 × g, 5 minutes), the supernatant was discarded and the pellet was resuspended and incubated (65 °C, 10 min) with DNA Releasing Reagent A (16 μL) and DNA Releasing Reagent B (16 μL). Protein Removal Reagent (8 μL) was added to the mixture which was then centrifuged (13,000 × g, 5 min). The supernatant was then added to a clean microcentrifuge tube and DNA was precipitated by adding isopropyl alcohol (24 μL). The mixture was centrifuged (13,000 × g, 10 min) and the pellet was washed by adding 70% ethanol (13,000 × g, 1 min). The supernatant was then removed by aspiration and the pellet was allied to dry inside a fume hood. TE buffer (10 mM Tris, 1 mM EDTA, 50 μL) was added to the pellet to afford the total isolated DNA. PCR analysis was performed using oligonucleotides listed in Supplementary Table 2.
Growth curves for yeast and cyanobacteria cells
Growth curve data were obtained by growing 200 uL of cells in sterile 96-well plates (Nunclon 167008). Yeast cells were grown aerobically with shaking at 30 °C and 240 rpm. Syn7942 cells were shaken at 30 °C and 70 rpm. Measurements were all obtained with the BioTek Synergy H1M Microplate Reader and Gen5 v.3.11.19 software. Pathlength correction for these measurements was determined manually. Water (200 μL) was added to a well and A975 and A900 were measured by plate reader. Water was also added to a quartz cuvette (Spectrocell R-3010-T) and the near-IR absorption spectrum was obtained using the Shimadzu UV-VIS-NIR Spectrophotometer UV-3600 and UVProbe v.2.34 software. Corrections to absorbance data were made with the following equation:
Analysis of yeast/cyanobacteria chimeras using pTIRF microscopy
Samples were prepared by extracting the chimeric cells from plates and washing them once with 1 M sorbitol. They were analyzed with a home-built TIRF microscope based on a Zeiss Axiovert 200 M stand. A Cobolt diode-pumped 561 nm laser was used in this work. The laser beam is combined with other two laser lines in the system by using a set of Semrock LaserMUX™ filters, and then sent through an acousto optic tunable filter (AOTF, Quanta Tech Inc). The AOTF is used through the microscope control software to simultaneously and independently control the power of each laser on sample. After the AOTF, a laser speckle-reducer (LSR-3005-17S-VIS, Optotune) is used with a set of achromatic lenses to provide homogeneous illumination across the entire field of view for TIRF or pTIRF microscopy. The dichroic beamsplitter used in the system is a Semrock LF405/488/561/635-B-000 and the emission filter is a Chroma bandpass filter HQ 653/95 nm. The pTIRF images were acquired with a Photometric 512 Evolve EMCCD camera. Samples were viewed and imaged using a 100X oil immersion objective lens with NA = 1.4. The software for microscope control and data acquisition was developed using C++ and Labview. All images were processed with Fiji, for example, to merge a qTIRF image and a brightfield image that were acquired from the same sample position. Fluorescence of chimeras was measured using ImageJ 1.53c. Images of chimeras excited with either 405 nm or 561 nm laser were concatenated and fluorescent regions of interest (ROIs) were identified by thresholding.
Analysis of yeast/cyanobacteria chimeras using fluorescence confocal microscopy
Stock solutions (40X) of Concanavalin A, Fluorescein Conjugate (ConA; Thermo Fisher C827) were prepared by dissolving the lyophilized powder in sodium bicarbonate (0.1 M) to a final concentration of 2 mg/mL. These solutions were stored at −20 °C without exposure to light. Directly prior to use in sample preparation, the stock solution was thawed and centrifuged for 10 s. Microscopy samples were prepared by extracting the chimeric cells from plates and washing them once with Hank’s Buffered Salt Solution (HBSS; NaCl (140 mM), KCl (5 mM), CaCl2 (1 mM), MgSO4 heptahydrate (0.4 mM), MgCl2 hexahydrate (0.5 mM), Na2PO4 dihydrate (0.3 mM), KH2PO4 (0.4 mM), D-glucose (6 mM), NaHCO3 (4 mM)). The cells were then incubated (37 °C, 10 min) in HBSS supplemented with ConA to a final concentration of 50 μg/mL, washed twice with HBSS, centrifuged and incubated with Karnovsky fixative (2% glutaraldehyde, 2.5% paraformaldehyde). Samples were analyzed with a commercial Leica SP8 fluorescence confocal microscope. Samples were viewed and imaged through a 63X/1.40 HC PL APO Oil CS2 lens and excited with 488 nm and 561 nm laser light. Emission wavelengths in the 510/20 nm range were detected with photomultiplier tube (PMT) detector, and emission wavelengths in the 650/20 range were detected using a high-sensitivity GaAsP HyD detector. Leica Application Suite (LASX) was used to collect raw data. All images were processed with Fiji to display an overlay of the two channels.
Analysis of yeast/cyanobacteria chimeras using TEM
Cells were extracted from agar plates in HBSS (700 μL) and pelleted by centrifugation (6000 × g, 5 min). The supernatant was removed and the cells were resuspended in Karnovsky fixative (20 μL) and incubated for 30 min. The cells were then pelleted and resuspended in HBSS. The medium was removed and replaced with a fixative (2.5% EM-grade glutaraldehyde and 2.0% EM-grade formaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4) for 3 h at 4 °C. The fixative was then removed, replaced briefly with buffer, and then replaced with 1% osmium tetroxide in buffer for 90 min. Each sample was then subjected to 10-minute buffer rinse, after which it was placed in 1% aqueous uranyl acetate and left overnight. The next day, each sample was dehydrated via a graded ethanol series, culminating in propylene oxide. Following a graded propylene oxide; Epon812 series, the nuclear pellets were embedded in Epon812 prior to cutting. Ultrathin (ca. 90 nm) Epon sections on grids were stained with 1% aqueous uranyl acetate and lead citrate62. After the grids dried, areas of interest were imaged at 160 kV, spot 3 using a Philips/FEI (now Thermo Fisher FEI) Tecnai G2 F20 S-TWIN transmission electron microscope in the Microscopy Suite at the Beckman Institute of Advanced Science and Technology (University of Illinois at Urbana-Champaign).
Growth dependence of chimeras on light
For partial light starvation after the introduction on cyanobacteria, SynJEC3 cells were introduced to yeast spheroplasts in the manner described, plated on and overlaid with Selection-I medium. The plates were incubated at 30 °C for 4 d inside a box covered in aluminum foil placed into the incubator. After this time, the contents of the overlaid agar plate were extracted by stabbing and plated on Selection-II medium grown either in 12 h day/night cycle or under the same conditions described to remove light. The growth of chimeras under these different conditions was quantified in the manner described. For complete light starvation, a flask containing BG-11 medium was inoculated with log-phase SynJEC3 cells to OD730 of 0.200 and incubated for 4 d without shaking inside a box covered in aluminum foil. These cells were introduced to yeast spheroplasts in the manner described, plated on Selection-I medium and incubated for 4 d at 30 °C inside a foil-covered box. After this time, the contents of the plate were extracted by stabbing the agar and plated on Selection-II medium. The plates were incubated for 4 d more in the absence of light.
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