Jun 26, 2019
Version 2
Biolistic Transformation of Amphidinium V.2
- Isabel Nimmo1,
- Ellen Nisbet2,
- Adrian Barbrook2,
- Chris Howe.2
- 1University of Cambridge;
- 2Department of Biochemistry, University of Cambridge
External link: https://doi.org/10.1038/s41592-020-0796-x
Protocol Citation: Isabel Nimmo, Ellen Nisbet, Adrian Barbrook, Chris Howe. 2019. Biolistic Transformation of Amphidinium. protocols.io https://dx.doi.org/10.17504/protocols.io.4r2gv8e
Manuscript citation:
Faktorová D, Nisbet RER, Robledo JAF, Casacuberta E, Sudek L, Allen AE, Ares M, Aresté C, Balestreri C, Barbrook AC, Beardslee P, Bender S, Booth DS, Bouget F, Bowler C, Breglia SA, Brownlee C, Burger G, Cerutti H, Cesaroni R, Chiurillo MA, Clemente T, Coles DB, Collier JL, Cooney EC, Coyne K, Docampo R, Dupont CL, Edgcomb V, Einarsson E, Elustondo PA, Federici F, Freire-Beneitez V, Freyria NJ, Fukuda K, García PA, Girguis PR, Gomaa F, Gornik SG, Guo J, Hampl V, Hanawa Y, Haro-Contreras ER, Hehenberger E, Highfield A, Hirakawa Y, Hopes A, Howe CJ, Hu I, Ibañez J, Irwin NAT, Ishii Y, Janowicz NE, Jones AC, Kachale A, Fujimura-Kamada K, Kaur B, Kaye JZ, Kazana E, Keeling PJ, King N, Klobutcher LA, Lander N, Lassadi I, Li Z, Lin S, Lozano J, Luan F, Maruyama S, Matute T, Miceli C, Minagawa J, Moosburner M, Najle SR, Nanjappa D, Nimmo IC, Noble L, Vanclová AMGN, Nowacki M, Nuñez I, Pain A, Piersanti A, Pucciarelli S, Pyrih J, Rest JS, Rius M, Robertson D, Ruaud A, Ruiz-Trillo I, Sigg MA, Silver PA, Slamovits CH, Smith GJ, Sprecher BN, Stern R, Swart EC, Tsaousis AD, Tsypin L, Turkewitz A, Turnšek J, Valach M, Vergé V, Dassow Pv, Haar Tvd, Waller RF, Wang L, Wen X, Wheeler G, Woods A, Zhang H, Mock T, Worden AZ, Lukeš J, Genetic tool development in marine protists: emerging model organisms for experimental cell biology. Nature Methods 17(5). doi: 10.1038/s41592-020-0796-x
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 26, 2019
Last Modified: June 26, 2019
Protocol Integer ID: 25114
Abstract
A method to transform the chloroplast of Amphidinium carterae using biolistics.
orcid.org/0000-0002-2469-0611
orcid.org/0000-0002-6975-8640
orcid.org/0000-0003-4487-196X
Materials
pAmpPSBA
gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 60
agattaagaa cgattagatt accttcctta ttattgggaa cgtttgaacg atgaattaaa 120
ctcacagtag gcccttcggg cgcctttcga acgacgtatt gacctaccta cctaagtaga 180
gatcgttgtg gcgacgtatc aatacccggg taaacaggta caactgttgt atggttatcc 240
tgcaacacct cttagatgct agaatgagtc cataaaggct tggaatctcc gcttgtagtt 300
atctctggtt aggtaatagt gcctttcctt ctttgagcct cctaccgaaa gtcaattcaa 360
cagcacagat gagtattatt aagagctcaa acaatgttga tcaaagacga tcgaaggata 420
cggaagcgta gagaatagac aatcgggaga ctatcattag tcctggacaa atgttgtcag 480
acatgaggtt gaccaggtca ttcataggct ctccggtcat tttgttccat ctctacccca 540
gtagagaaaa atccaggtca tatcatagga gatggaactg agagatcgag agaacgaaga 600
cagaaaggag gtgaaaggta gttggcatgt tgatctggcg agtcagaggc atcaaacagg 660
aacgaaggta atagaatgaa tgataattga tgaaacgaca atgaaagtcc tctaaagaag 720
aggcaactaa taaagatgaa acatatctcg ggggtgcgcc taagtcaggc taagaaagca 780
tcaaacagat acaaccgctg ggcaggtcaa caagtgatta atcctgatct aaggatgatt 840
acgacctgcg gaaaggtggt tacaatacct catacctgta tgactgatcg gtgctttaat 900
cacaccttgc aagttgccaa ttacattgag taggcatctt taatagcttg tagggttcac 960
accgcagtct aaaggtagaa aggtccaaca atcaggtcat gtggcccaga aaccacgctg 1020
gtaacctgag accctacctc ccttcgggga tcctatcaaa tggtctgtgt attcagtccc 1080
cgtcttacgg agtgcttgtg gcggtggagt ggcaggggta ggcaagtagt cgagtggtga 1140
gctgtaatgg tgtatccgcg tttaccttcc tggtacctcg gtgcctcggt aagaagacga 1200
actctcgtta gtgaggccag cgtaaacctt tttccatatc tattatgaca agccttattc 1260
gctctaactc ctggggttct ttcgttcaaa caatcacttc ttcctctaac cgtctttata 1320
tcggttggtt cggtctcctc gtcttcccac ttctctccct tgctaccgta gcttatatca 1380
ctgctttctt ccttgctcct gcagtcgata ttgatggtat ccgtgagcca gttgcaggtt 1440
cccttatcta tggtaacaac atcatctctg gtgctgttat tccttcttct aacgcaatcg 1500
gtgtccattt ctatcctctc tgggaatccc ttggtcttga tgaatggctc tacaacggtg 1560
gtacttatca gttcgttgtg ttccacttct tcctcggtgt ctgcggttgg atgggtcgtg 1620
aatgggaatt ctcctatcgt ctcggtatgc gtccatggat cttcgtagca ttctccgctc 1680
caattgcggc agcagctgca gtgttcatta tttacccaat cggtcagggt tccttctccg 1740
atggtatgcc acttggtatc cagggtactt tcaacttcat gcttgtcttc caagctgagc 1800
acaagatcct catgcatcct ttccacattc ttggtgtagc aggtgtcttc ggtggttctc 1860
tcttctccgc aatgcacggt tctcttgtat cctcttctct ccttgcagag actgctggtt 1920
ctgagtccct taacaacggt tacgtattcg gtcaagagga tgagacttat tccatctctg 1980
cagcacacgc atatttcggt cgtcttatct tccagtatgc tggcttcaac aactcccgtt 2040
ctcttcactt cttccttgca gcttggcctg taattggtat ctggttcacc tctcttggtg 2100
ttgctactat ggcattcaac cttaacggtt tcaacttcaa ccagtctatt cttgacgaat 2160
ctggtcacta cattaactct tgggctgata tcctcaaccg tgctgatctt ggtatcgagg 2220
taatgcacga gcgtaacgct cataacttcc ctcttgatct tgcatagaat caaccccttt 2280
atccctctct ttgggactct tagaacgact aggctttctg attaacgatg atgtaatatt 2340
attaagaacg atggctagta ataatgcacg aatcactagt gaattcgcgg ccgcctgcag 2400
gtcgaccata tgggagagct cccaacgcgt tggatgcata gcttgagtat tctatagtgt 2460
cacctaaata gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 2520
ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 2580
tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 2640
ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 2700
gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 2760
gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 2820
ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 2880
ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 2940
cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 3000
ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 3060
tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 3120
gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 3180
tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 3240
gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 3300
tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 3360
ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 3420
agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 3480
gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 3540
attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 3600
agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 3660
atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 3720
cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 3780
ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 3840
agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 3900
tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 3960
gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 4020
caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 4080
ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 4140
gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 4200
tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 4260
tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 4320
cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 4380
cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 4440
gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 4500
atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 4560
agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 4620
ccccgaaaag tgccacctga tgcggtgtga aataccgcac agatgcgtaa ggagaaaata 4680
ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa 4740
atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa 4800
tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 4860
gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 4920
ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct 4980
aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 5040
gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc 5100
gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat tcgccattca 5160
ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg 5220
cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac 5280
gacgttgtaa aacgacggcc agtgaattgt aatacgactc actata 5326
To be completed in advance
To be completed in advance
Prepare artificial minicircle (pAmpPSBA) at 1mg ml-1. pAmpPSBA contains a bacterial origin of replication and an ampicillin resistance marker, so transformation and growth using a suitable E. coli strain, followed by a miniprep, is the simplest method to achieve this. pAmpPSBA also contains a linearised minicircle containing the core region and a modified form of PSBA to confer resistance to the herbicide Atrazine.
Grow wild type Amphidinium carterae in f/2 medium to early log-phase.
Prepare f/2 plates (1.5% agarose in f/2 medium) and leave to dry for 3-4 hours before storing at 4ºC. (This drying step is not strictly necessary, but allows the cells to be transferred to the plates much more quickly, which reduces the subsequent time between plating and shooting).
Autoclave stopping screens, macrocarriers and 1,550 PSI rupture disks (all supplied by BioRad).
Cell Preparation
Cell Preparation
Count the cells. For each plate to be transformed (including necessary controls), between 1-5x107 cells are needed. Harvest an appropriate volume for the number of plates to be shot.
Centrifuge the cells at low g (~1,500g) and resuspend the pellet in a very small volume (~500µl) of fresh f/2 medium.
Spot the cells onto the centre of the f/2 plate dropwise. Leave to dry. Cells should for a circle of no more than 2cm diameter to maximise the number of cells in the region bombarded by gold particles. Note that Amphidinium does not survive long term on solid plates, so aim to shoot as soon after drying as practical.
DNA Preparation
DNA Preparation
DNA Precipitation is carried out using Seashell Technology’s “DNAdelTM gold carrier” delivery system. 550nm gold particles are used throughout.
Sonicate the DNAdelTM gold particles (supplied at 50mg ml-1) to dissociate aggregates. 1-2 minutes should normally be sufficient, but the presence of aggregates can be detected by eye.
Add artificial minicircle to gold particles at a ratio of 2.5µg DNA per mg gold. Using very concentrated DNA stock keeps volume low and allows for high concentrations of DNA and gold to maximise DNA precipitation onto the gold particles.
DNA Preparation
DNA Preparation
Vortex the DNA-gold particle mixture briefly.
Centrifuge at 10,000g for 10s to pellet the gold particles.
Remove the supernatant and add 500µl ice cold 100% ethanol. Vortex briefly.
Centrifuge at 10,000g for 10s to pellet the gold particles.
Remove the supernatant. Add 10µl ice cold 100% ethanol for each 0.5mg gold in the preparation. (e.g. add 30µl for the 1.5mg gold prepared for shooting 3 plates).
Briefly sonicate the solution to minimise aggregation and allow for reliable delivery. Again, 1-2 minutes will normally be sufficient, but sonication should continue untill even suspension of the gold particles is achieved.
Bombardment Preparation
Bombardment Preparation
Wash the macrocarriers in 70% ethanol and allow to dry.
Wash each rupture disk and macrocarrier in isopropanol and allow to dry.
Transfer a 10µl aliquot of the prepared gold particles (see above) to the centre of each macrocarrier. Allow to dry.
Bombardment
Bombardment
Bombardment is carried out in a Biorad ‘Biolistic PDS-1000/He’ device using Biorad stopping screens, macrocarriers and rupture disks.
Shooting is carried out using 1,550 PSI rupture disks. (These show higher rates of transformation that 1,100 or 1,350 PSI disks.)
Shooting is carried out in vacuum at ≥25 in. Hg.
Plates for transformation are placed in the middle slot in the Biolistics device (i.e. the highest possible position for a plate. Higher slots are occupied by the rupture disk and macrocarrier).
Post Shooting
Post Shooting
Cells should be resuspended as soon as possible after shooting to minimise cell death.
Resuspend all cells from each plate in 5ml fresh f/2 medium. As many cells as possible should be washed off the surface of the plate and collected in the desired vessel for growth.
Leave to recover overnight.
Dilute the cells as and apply selection criteria. (e.g. each plate's cells are diluted to a final volume of 50ml with 2ug ml-1 Atrazine).
Monitor growth by microscopy every 1-2 days. Untransformed Amphidinium carterae are typically dead at 10-12 days post-application of Atrazine.