An inducible expression system in the alga Nannochloropsis gaditana controlled by the nitrate reductase promoter Journal of Applied Phycology 1 2 3 1 Harry O. Jackson ; Adokiye Berepiki ; Alison J. Baylay1; Matthew J. Terry ; C. Mark Moore 1 & Thomas S. Bibby *
1
Ocean and Earth Sciences, National Oceanography Centre, University of Southampton, Waterfront Campus, European Way, Southampton, SO14 3ZH, United Kingdom 2 Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, United Kingdom 3 Biological Sciences Faculty of Natural & Environmental Sciences, Life Sciences Building 85, University of Southampton, Highfield Campus, Southampton, SO17 1BJ, United Kingdom Corresponding author*:
[email protected]
Online Resource 1
Online Resource 1. Nucleotide sequences of primers used to construct expression cassettes and for genotyping of transformants. Nucleotides acting as extensions to guide recombination are shown in uppercase.
Primer name
Nucleotide sequence (5ʹ-3ʹ)
PNR fw XX ext
GGAAGGGCGATCGGTGCGGGCCGTTTAAACggcttctcacgtcgttctcatc
PNR rv eGFP ext
GGTGAACAGCTCCTCGCCCTTGCTCACCATcgaccaccctggagtga
PLCP fw XX ext
GGAAGGGCGATCGGTGCGGGCCGTTTAAACcgcattcaggtctagtaag
PLCP rv eGFP ext
GGTGAACAGCTCCTCGCCCTTGCTCACCATtattgcttctgttcaaatgt
eGFP fw
atggtgagcaagggcgagga
eGFP rv
tcacttgtacagctcgtcca
TATPα fw eGFP ext
ACTCTCGGCATGGACGAGCTGTACAAGTGAtagcggcgcttttgacc
TATPα rv CC ext
TTGTGTCATGAATTAACAGTTAACGAATACggacagggatacctgtgttt
PUEP fw CC ext
GTATTCGTTAACTGTTAATTCATGACACAAcatcctgctgtatgattttggcacaacg
PUEP rv hph ext
CGTCGCGGTGAGTTCAGGCTTTTTACCCATagctgctgccccgaccgtatc
hph fw
atgggtaaaaagcctgaactcaccg
hph rv
ttattcctttgccctcggacgagtgc
TNOS fw hph ext
CCCAGCACTCGTCCGAGGGCAAAGGAATAAtgataacagcgcttagagctc
TNOS rv YY ext
TGGAATTGTGAGCGGATAACAAGTTTAAACcagaaataggcgcgcctaattc
YY fw PmeI ext
gtttaaacttgttatccgctcacaattccacac
XX rv PmeI ext
gtttaaacggcccgcaccgatc
Online Resource 2
a 9000
2.5
8000
2
7000 6000
A540 nm
Relative fluorescence units (RFU)
b
5000 4000 3000 2000
1.5 1 0.5
1000 0 0
2
4
6
ng
µL-1
8
eGFP
10
12
0 0.00E+00
5.00E+07 cells mL-1
1.00E+08
Online Resource 2. Standard curves used for calculation of eGFP expression (ng eGFP per 107 cells). (a) Plot of ng/µL eGFP versus detector response in relative fluorescence units (RFU) for generation of standard curve for conversion of eGFP fluorescence levels (RFU/Abs- WT subtracted) to ng/µL eGFP. (b) Plot of N. gaditana cells/mL vs OD540 for generation of standard curve for conversion of absorbance values at 540 nm to cells/mL
Online Resource 3
Genbank format annotated vector map for pNR. LOCUS DEFINITION ACCESSION VERSION SOURCE ORGANISM COMMENT COMMENT COMMENT
pNR_
10175 bp
DNA
circular
17/03/2018
Serial Cloner Genbank Format SerialCloner_Type=DNA SerialCloner_Comments=Homologous recombination reaction between [pNR hph] as a tar get sequence and [XX-pNR vector cassette-YY] as an inserted sequence. COMMENT SerialCloner_Ends=0,0,,0, FEATURES Location/Qualifiers misc_feature complement(8365..8387) /label=pGEX_3_primer /SerialCloner_Color=&hDC143C /SerialCloner_Show=False /SerialCloner_Protect=True /SerialCloner_Arrow=True /SerialCloner_Desc=label="pGEX_3_primer" - complement(29..51) \ promoter 8538..8751 /label=URA3_promoter /SerialCloner_Color=&hF08080 /SerialCloner_Show=True /SerialCloner_Protect=True /SerialCloner_Arrow=True /SerialCloner_Desc=label="URA3_promoter" - 202..415 \ gene 8753..9553 /label=URA3_URA3 /SerialCloner_Color=&hFF4500 /SerialCloner_Show=False /SerialCloner_Protect=True /SerialCloner_Arrow=True /SerialCloner_Desc=label="URA3" - gene="URA3" - 417..1217 \ CDS 8753..9556 /label=ORF frame 3_URA3_URA3 /SerialCloner_Color=&hFF4500 /SerialCloner_Show=True /SerialCloner_Protect=True /SerialCloner_Arrow=True /SerialCloner_Desc=label="URA3" - gene="URA3" - note="ORF frame 3" translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKEL LELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVK LQYSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLS - TGEYTKGTVDIAKSDKDFVIGFI \ rep_origin complement(9819..10125) /label=f1_origin /SerialCloner_Color=&hF08080 /SerialCloner_Show=True /SerialCloner_Protect=True /SerialCloner_Arrow=True /SerialCloner_Desc=label="f1_origin" - complement(1483..1789) \ misc_feature complement(10158..24) /label=lacZ_a [5'] /SerialCloner_Color=&hDC143C /SerialCloner_Show=False /SerialCloner_Protect=True /SerialCloner_Arrow=True /SerialCloner_Desc=label="lacZ_a" - complement(1822..1965) \ misc_feature complement(4795..24) /label=M13_pUC_rev_primer [3'] [5'] [3'] /SerialCloner_Color=&hDC143C /SerialCloner_Show=False /SerialCloner_Protect=True /SerialCloner_Arrow=True /SerialCloner_Desc=label="M13_pUC_rev_primer" - complement(2176..2198) \ rep_origin complement(5160..5779) /label=pBR322_origin
-
/SerialCloner_Color=&hF08080 /SerialCloner_Show=True /SerialCloner_Protect=True /SerialCloner_Arrow=True /SerialCloner_Desc=label="pBR322_origin" - complement(2550..3169) \ CDS complement(5934..6794) /label=ORF frame 3_Ampicillin_Ampicillin /SerialCloner_Color=&hFF4500 /SerialCloner_Show=False /SerialCloner_Protect=True /SerialCloner_Arrow=True /SerialCloner_Desc=label="Ampicillin" - gene="Ampicillin" - note="ORF frame 3" translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL - DRWEPELNEAI \ gene complement(5934..6794) /label=Ampicillin_Ampicillin /SerialCloner_Color=&hFF4500 /SerialCloner_Show=True /SerialCloner_Protect=True /SerialCloner_Arrow=True /SerialCloner_Desc=label="Ampicillin" gene="Ampicillin" complement(3324..4184) \ promoter complement(6836..6864) /label=AmpR_promoter /SerialCloner_Color=&hF08080 /SerialCloner_Show=True /SerialCloner_Protect=True /SerialCloner_Arrow=True /SerialCloner_Desc=label="AmpR_promoter" - complement(4226..4254) \ rep_origin complement(6926..7897) /label=2micron2_origin /SerialCloner_Color=&hF08080 /SerialCloner_Show=True /SerialCloner_Protect=True /SerialCloner_Arrow=True /SerialCloner_Desc=label="2micron2_origin" - complement(4316..5287) \ misc_feature complement(7889..7936) /label=FRT /SerialCloner_Color=&hDC143C /SerialCloner_Show=False /SerialCloner_Protect=True /SerialCloner_Arrow=True /SerialCloner_Desc=label="FRT" - complement(5279..5326) \ misc_feature 2..31 /label=XX extension /SerialCloner_Color=&h84A4C0 /SerialCloner_Show=True /SerialCloner_Protect=True /SerialCloner_Arrow=True misc_feature 32..1350 /label=PNR /SerialCloner_Color=&h0000FF /SerialCloner_Show=True /SerialCloner_Protect=True /SerialCloner_Arrow=True misc_feature 1351..2070 /label=egfp /SerialCloner_Color=&h00FF00 /SerialCloner_Show=True /SerialCloner_Protect=True /SerialCloner_Arrow=True misc_feature 2071..2578 /label=TATPa /SerialCloner_Color=&hFF0000 /SerialCloner_Show=True /SerialCloner_Protect=True /SerialCloner_Arrow=True misc_feature 2579..2608 /label=CC extension /SerialCloner_Color=&h84A4C0 /SerialCloner_Show=True /SerialCloner_Protect=True /SerialCloner_Arrow=True misc_feature 2609..3301 /label=PUEP /SerialCloner_Color=&h0000FF
misc_feature
misc_feature
misc_feature
/SerialCloner_Show=True /SerialCloner_Protect=True /SerialCloner_Arrow=True 3302..4330 /label=hph /SerialCloner_Color=&hFF7F00 /SerialCloner_Show=True /SerialCloner_Protect=True /SerialCloner_Arrow=True 4331..4788 /label=TNOS /SerialCloner_Color=&hFF0000 /SerialCloner_Show=True /SerialCloner_Protect=True /SerialCloner_Arrow=True 4789..4818 /label=YY extension /SerialCloner_Color=&h84A4C0 /SerialCloner_Show=True /SerialCloner_Protect=True /SerialCloner_Arrow=True
ORIGIN 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241
gGGAAGGGCG ttctccccct atttctatgt tcaacacgga cagggaaata catggagggt gtcactgcga tatttcttgc catttctgca aatttctctg attgatacct agagcgcaaa gggcgtgaac gtgaacaacc ttaattgccg aataatgacg ttttacgcca ggggaagcgc acactggtgc ctcggattcc aattatgcat tgctcacgcg aacatttctc ggggtggtgc tccggcgagg accggcaagc tgcttcagcc gaaggctacg gccgaggtga ttcaaggagg gtctatatca aacatcgagg gacggccccg gaccccaacg actctcggca tttaatgtat ccacatccgt cttctccatt ggtcatgccc tgtaagtctt tacaaagtcc gatttatagc ttccgctggc attcgttaac ggttggaggg aaaaaaggaa aacaagaggg cacaaagcgt cccaaccaca tgcatccggg gagcttgggc ggtcacaccc aacataccca gttaatgatc gagtttttaa
ATCGGTGCGG ttccttttct ggaggctgac ttgagagagc ggttggaagg gaagataagc agttgacctg acatcttgaa tacgtcataa aatggttagc ccactaagga gcttggaata aaccacgatc actataagaa cataatctga ccattacttg taactgcgtt ggtagatttt catgtatggg gtgttaggcg catgcgactt acttcatgaa acatcactcc ccatcctggt gcgagggcga tgcccgtgcc gctaccccga tccaggagcg agttcgaggg acggcaacat tggccgacaa acggcagcgt tgctgctgcc agaagcgcga tggacgagct tcgaaacacg cctcacgtac ttactttttg tttatgggca caaaaaagaa tctacaattc tttgcatgat acctcattat tgttaattca tgagggagat cacacgcgcc acaccacgtc cggaaaggga cacattcctc gcaggtcgac atttctttac gcgatcgaga tcgttaagtc gggtacgttt cgcaactgaa
GCCGTTTAAA tccgtcgttt gttttccggg actgtacgca ggagcaattc tcgctctgaa aaagcgtgtt gatagtctat ttccacgatg actttccttt gtcaaatcaa atctctttat agagtttagg ttgaagaggc ccgtcgtctg aagtttcgcg tccccgttct gttttcaagg actattctga tcgtaacgaa gtgtgaatgt tatgtcttcc agggtggtcg cgagctggac tgccacctac ctggcccacc ccacatgaag caccatcttc cgacaccctg cctggggcac gcagaagaac gcagctcgcc cgacaaccac tcacatggtc gtacaagtga ctttttaaga ggtgtgatgt tgtttctgtc cgacggcagg gaaagcgggg tacagtccta cgtctccgag gcgatacgca tgacacaaca ggtggtgaga caggatagaa ggtgaagagg ggcgaccgct ccgcgaggct gagccagcat cggacacgtc aagcagagag tgatgatggc aaaggcgtgg gatttcatgt
Cggcttctca ctcccacaga tcagcgccga gtgcagagcg gcagaaggaa agtaaatctg tttcaaatcg acgcaggatg actagcaatg gtttttttgt tagctttgat atgtcccatt agagcgatca gatcctccaa gatagcctgg gggctttttt ccaagggctc aaggaccaag cggatctccg cattaggaag gcatcaatct cttacatact atggtgagca ggcgacgtaa ggcaagctga ctcgtgacca cagcacgact ttcaaggacg gtgaaccgca aagctggagt ggcatcaagg gaccactacc tacctgagca ctgctggagt tagcggcgct tctcaaatga ttttttaatt ttcacagtct attttggttt acggaaactt gttgttcacg catttcagaa ctccatataa tcctgctgta catcagaaat ccaggaccac tgcgtgagac tcgctggagg caagtgccat ggtttggtcg gcttcttaca tcggaccccc tggtgcaaag gcaaggttat ctgacttgga
cgtcgttctc acaattacgg tcggacgatg tggtaggaca agctcagcga acaaatcaag tcgcccaggc aaaaggagga tggaatagat cctatgaaat cacaagcaga gattaacatc actccaagca gaacgctgcc cagacgggaa tcctggctcg cgccgtcggc ggtgtgaggt ccgcgcaact acatcggatc tttgcctgtt cctcctgatc agggcgagga acggccacaa ccctgaagtt ccctgaccta tcttcaagtc acggcaacta tcgagctgaa acaactacaa tgaacttcaa agcagaacac cccagtccgc tcgtgaccgc tttgaccggg aacatgtatc ttttatatga ttctctgcga ttgaaagaat cttgcttgtg cgacttcacg tgctataata acacaggtat tgattttggc ggagagaata attccaagtc agaggacacc ggggccgtcc gccgccgagt ccgacaacgc tttttacatt ttcaagcagg ccaaggcagc ttaacgctgg gatacggtcg
atcgtctgct gagatgctgg attgaactct tagaatcgcg tcgagaaaaa cacagatgag gaagtgttat tcaactttgc cttccttttg ttaaatggta agaaatgata tatcctacaa ctaaaagggc cgaccaggca aacttcatgg gaattatact aactttgtat ggcttttgaa taactcccgc ttagatgaga gcattacgca acgacaacac gctgttcacc gttcagcgtg catctgcacc cggcgtgcag cgccatgccc caagacccgc gggcatcgac cagccacaac gatccgccac ccccatcggc cctgagcaaa cgccgggatc aactcacgca cgtatgctct aagtgtaagg gctgtgagag ttctgtcggc tgttatacat cagcagacca tggaatgatc ccctgtccgt acaacgacgc acggtagaga cctcaccagc agacaacgtg acagcctgcg gccatgctcc agatttgatg ccaattcaga aaaggcaagc cagtcgatat tagcatcgta gggcagcagc
3301 3361 3421 3481 3541 3601 3661 3721 3781 3841 3901 3961 4021 4081 4141 4201 4261 4321 4381 4441 4501 4561 4621 4681 4741 4801 4861 4921 4981 5041 5101 5161 5221 5281 5341 5401 5461 5521 5581 5641 5701 5761 5821 5881 5941 6001 6061 6121 6181 6241 6301 6361 6421 6481 6541 6601 6661 6721 6781 6841 6901 6961 7021 7081 7141 7201 7261 7321 7381 7441 7501 7561 7621 7681 7741 7801 7861
tatgggtaaa cgacagcgtc cgatgtagga agatcgttat cattggggaa gttgcaagac ggatgcgatc aggaatcggt gtatcactgg tgagctgatg cggctccaac ggcgatgttc ggcttgtatg gccgcggctc tgacggcaat cggagccggg tggctgtgta aaaggaataa aataaagttt tgttgaatta gggtttttat agcgcgcaaa tggttccgga gctcacaatt atgagtgagc TATCCGCTCA gcctaatgag ggaaacctgt cgtattgggc cggcgagcgg aacgcaggaa gcgttgctgg tcaagtcaga agctccctcg ctcccttcgg taggtcgttc gccttatccg gcagcagcca ttgaagtggt ctgaagccag gctggtagcg caagaagatc taagggattt aaatgaagtt tgcttaatca tgactccccg gcaatgatac gccggaaggg aattgttgcc gccattgcta ggttcccaac tccttcggtc atggcagcac ggtgagtact ccggcgtcaa ggaaaacgtt atgtaaccca gggtgagcaa tgttgaatac ctcatgagcg acatttcccc atgcaacgcg gaaatgcaac caaaaatgca gaacagaaat ttctacaaaa tttctccttt taaggttaga cacttcccgc atccccgatt gcgttgatga atactacgta tcttactaca gtcgagttta agcacagaga aatattttag gagcgctttt
aagcctgaac tccgacctga gggcgtggat gtttatcggc ttcagcgaga ctgcctgaaa gctgcggccg caatacacta caaactgtga ctttgggccg aatgtcctga ggggattccc gagcagcaga cgggcgtata ttcgatgatg actgtcgggc gaagtactcg tgataacagc cttaagattg cgttaagcat gattagagtc ctaggataaa accaattcgt ccacacaaca taactcacat CAATTCCAca tgaggtaact cgtgccagct gctcttccgc tatcagctca agaacatgtg cgtttttcca ggtggcgaaa tgcgctctcc gaagcgtggc gctccaagct gtaactatcg ctggtaacag ggcctaacta ttaccttcgg gtggtttttt ctttgatctt tggtcatgag ttaaatcaat gtgaggcacc tcgtgtagat cgcgagaccc ccgagcgcag gggaagctag caggcatcgt gatcaaggcg ctccgatcgt tgcataattc caaccaagtc tacgggataa cttcggggcg ctcgtgcacc aaacaggaag tcatactctt gatacatatt gaaaagtgcc agagcgctaa gcgaaagcgc acgcgagagc gcaacgcgag atgcatcccg gtgcgctcta agaaggctac gtttactgat atattctata ttcttcattg taggaaatgt atttttttgt gatgcaagtt tatatagcaa tagctcgtta ggttttcaaa
tcaccgcgac tgcagctctc atgtcctgcg actttgcatc gcctgaccta ccgaactgcc atcttagcca catggcgtga tggacgacac aggactgccc cggacaatgg aatacgaggt cgcgctactt tgctccgcat cagcttgggc gtacacaaat ccgatagtgg gcttagagct aatcctgttg gtaataatta ccgcaattat ttatcgcgcg aatcatggtc tacgagccgg taggctgaat caacatagga cacattaatt gcattaatga ttcctcgctc ctcaaaggcg agcaaaaggc taggctccgc cccgacagga tgttccgacc gctttctcat gggctgtgtg tcttgagtcc gattagcaga cggctacact aaaaagagtt tgtttgcaag ttctacgggg attatcaaaa ctaaagtata tatctcagcg aactacgata acgctcaccg aagtggtcct agtaagtagt ggtgtcacgc agttacatga tgtcagaagt tcttactgtc attctgagaa taccgcgcca aaaactctca caactgatct gcaaaatgcc cctttttcaa tgaatgtatt acctgaacga tttttcaaac tattttacca gctaattttt agcgctattt agagcgctat taatgcagtc tttggtgtct tactagcgaa ccgatgtgga gtcagaaaat ttacattttc ctaaagagta caaggagcga agagatactt cagtccggtg agcgctctga
gtctgtcgag ggagggcgaa ggtaaatagc ggccgcgctc ttgcatctcc cgctgttctg gacgagcggg tttcatatgc cgtcagtgcg cgaagtccgg ccgcataaca cgccaacatc cgagcggagg tggtcttgac gcagggtcga cgcccgcaga aaaccgacgc cgaatttccc ccggtcttgc acatgtaatg acatttaata cggtgtcatc atagctgttt aagcataaag taggcgcgcc gccggaagca gcgttgcgct atcggccaac actgactcgc gtaatacggt cagcaaaagg ccccctgacg ctataaagat ctgccgctta agctcacgct cacgaacccc aacccggtaa gcgaggtatg agaaggacag ggtagctctt cagcagatta tctgacgctc aggatcttca tatgagtaaa atctgtctat cgggagggct gctccagatt gcaactttat tcgccagtta tcgtcgtttg tcccccatgt aagttggccg atgccatccg tagtgtatgc catagcagaa aggatcttac tcagcatctt gcaaaaaagg tattattgaa tagaaaaata agcatctgtg aaagaatctg acgaagaatc caaacaaaga taccaacaaa ttttctaaca tcttgataac attttctctt gctgcgggtg ttgcgcatac tatgaacggt gtattgtttt atactagaga aaggtggatg ttgagcaatg cgtttttggt agttcctata
aagtttctga gaatctcgtg tgcgccgatg ccgattccgg cgccgtgcac cagccggtcg ttcggcccat gcgattgctg tccgtcgcgc cacctcgtgc gcggtcattg ttcttctgga catccggagc caactctatc tgcgacgcaa agcgcggccg cccagcactc cgatcgttca gatgattatc catgacgtta cgcgatagaa tatgttacta cctgtgtgaa tgtaaagcct tatttctgGT taaagtgtaa cactgcccgc gcgcggggag tgcgctcggt tatccacaga ccaggaaccg agcatcacaa accaggcgtt ccggatacct gtaggtatct ccgttcagcc gacacgactt taggcggtgc tatttggtat gatccggcaa cgcgcagaaa agtggaacga cctagatcct cttggtctga ttcgttcatc taccatctgg tatcagcaat ccgcctccat atagtttgcg gtatggcttc tgtgcaaaaa cagtgttatc taagatgctt ggcgaccgag ctttaaaagt cgctgttgag ttactttcac gaataagggc gcatttatca aacaaatagg cttcattttg agctgcattt tgtgcttcat atctgagctg gaatctatac aagcatctta tttttgcact ccataaaaaa cattttttca tttgtgaaca ttcttctatt cgattcactc taaacataaa ggtaggttat tttgtggaag tttttgaaag ctttctagag
tcgaaaagtt ctttcagctt gtttctacaa aagtgcttga agggtgtcac cggaggccat tcggaccgca atccccatgt aggctctcga acgcggattt actggagcga ggccgtggtt ttgcaggatc agagcttggt tcgtccgatc tctggaccga gtccgagggc aacatttggc atataatttc tttatgagat aacaaaatat gatcgggaat attgttatcc ggggtgccta TTAAACTTGT agcctggggt tttccagtcg aggcggtttg cgttcggctg atcaggggat taaaaaggcc aaatcgacgc tccccctgga gtccgccttt cagttcggtg cgaccgctgc atcgccactg tacagagttc ctgcgctctg acaaaccacc aaaaggatct aaactcacgt tttaaattaa cagttaccaa catagttgcc ccccagtgct aaaccagcca ccagtctatt caacgttgtt attcagctcc agcggttagc actcatggtt ttctgtgact ttgctcttgc gctcatcatt atccagttcg cagcgtttct gacacggaaa gggttattgt ggttccgcgc tagaacaaaa ttacagaaca ttttgtaaaa catttttaca ttcttttttg gattactttt gtaggtccgt agcctgactc agataaaggc gaaagtgata ttgtctctat tatgaatagt aaatgtagag atagggatat cggtattcgc tgcgtcttca aataggaact
7921 7981 8041 8101 8161 8221 8281 8341 8401 8461 8521 8581 8641 8701 8761 8821 8881 8941 9001 9061 9121 9181 9241 9301 9361 9421 9481 9541 9601 9661 9721 9781 9841 9901 9961 10021 10081 10141 //
tcggaatagg tgcgcacata tatacatgag atttatgtag gtatcgtatg tggattagtc cattattatc gcgtttcggt ttgtctgtaa cgggtgtcgg taccacagct tctttgaaat cagacttaga attcttaacc agctacatat tatcatgcac attactggag tatcttgact gtacaatttt gcagtactct tgtggtgggc acctagaggc atatactaag tgctcaaaga tgtgggttta ggtctctaca tgctaaggta cggccagcaa tagagcttca gcgtaaggag gttaaatttt ttataaatca tccactatta tggcccacta actaaatcgg cgtggcgaga agcggtcacg gtcgcgccat
aacttcaaag cagctcactg aagaacggca gatgaaaggt cttccttcag tcatccttca atgacattaa gatgacggtg gcggatgccg ggctggctta tttcaattca ttttttgatt ttggtatata caactgcaca aaggaacgtg gaaaagcaaa ttagttgaag gatttttcca ttactcttcg gcgggtgtat ccaggtattg cttttgatgt ggtactgttg gacatgggtg gatgacaagg ggatctgaca gagggtgaac aactaaaaaa atttaattat aaaataccgc tgttaaatca aaagaataga aagaacgtgg cgtgaaccat aaccctaaag aaggaaggga ctgcgcgtaa tcgccattca
cgtttccgaa ttcacgtcgc tagtgcgtgt agtctagtac cactaccctt atgctatcat cctataaaaa aaaacctctg ggagcagaca actatgcggc attcatcatt cggtaatctc tacgcatatg gaacaaaaac ctgctactca caaacttgtg cattaggtcc tggagggcac aagacagaaa acagaatagc ttagcggttt tagcagaatt acattgcgaa gaagagatga gagacgcatt ttattattgt gttacagaaa ctgtattata atcagttatt atcaggaaat gctcattttt ccgagatagg actccaacgt caccctaatc ggagcccccg agaaagcgaa ccaccacacc ggctgcgcaa
aacgagcgct acctatatct ttatgcttaa ctcctgtgat tagctgttct ttcctttgat taggcgtatc acacatgcag agcccgtcag atcagagcag ttttttttat cgaacagaag tagtgttgaa ctgcaggaaa tcctagtcct tgcttcattg caaaatttgt agttaagccg atttgctgac agaatgggca gaagcaggcg gtcatgcaag gagcgacaaa aggttacgat gggtcaacag tggaagagga agcaggctgg agtaaatgca accctatgcg tgtaaacgtt taaccaatag gttgagtgtt caaagggcga aagttttttg atttagagct aggagcgggc cgccgcgctt ctgtt
tccgaaaatg gcgtgttgcc atgcgtactt attatcccat atatgctgcc attggatcat acgaggccct ctcccggaga ggcgcgtcag attgtactga tctttttttt gaagaacgaa gaaacatgaa cgaagataaa gttgctgcca gatgttcgta ttactaaaaa ctaaaggcat attggtaata gacattacga gcagaagaag ggctccctat gattttgtta tggttgatta tatagaaccg ctatttgcaa gaagcatatt tgtatactaa gtgtgaaata aatattttgt gccgaaatcg gttccagttt aaaaccgtct gggtcgaggt tgacggggaa gctagggcgc aatgcgccgc
caacgcgagc tgtatatata atatgcgtct tccatgcggg actcctcaat actaagaaac ttcgtctcgc cggtcacagc cgggtgttgg gagtgcacca gatttcggtt ggaaggagca attgcccagt tcatgtcgaa agctatttaa ccaccaagga cacatgtgga tatccgccaa cagtcaaatt atgcacacgg taacaaagga ctactggaga tcggctttat tgacacccgg tggatgatgt agggaaggga tgagaagatg actcacaaat ccgcacagat taaaattcgc gcaaaatccc ggaacaagag atcagggcga gccgtaaagc agccggcgaa tggcaagtgt tacagggcgc
Online Resource 4
> 1-1319, PNR GGCTTCTCACGTCGTTCTCATCGTCTGCTTTCTCCCCCTTTCCTTTTCTTCCGTCGTTTCTCCCACAGAA CAATTACGGGAGATGCTGGATTTCTATGTGGAGGCTGACGTTTTCCGGGTCAGCGCCGATCGGACGATGA TTGAACTCTTCAACACGGATTGAGAGAGCACTGTACGCAGTGCAGAGCGTGGTAGGACATAGAATCGCGC AGGGAAATAGGTTGGAAGGGGAGCAATTCGCAGAAGGAAAGCTCAGCGATCGAGAAAAACATGGAGGGTG AAGATAAGCTCGCTCTGAAAGTAAATCTGACAAATCAAGCACAGATGAGGTCACTGCGAAGTTGACCTGA AAGCGTGTTTTTCAAATCGTCGCCCAGGCGAAGTGTTATTATTTCTTGCACATCTTGAAGATAGTCTATA CGCAGGATGAAAAGGAGGATCAACTTTGCCATTTCTGCATACGTCATAATTCCACGATGACTAGCAATGT GGAATAGATCTTCCTTTTGAATTTCTCTGAATGGTTAGCACTTTCCTTTGTTTTTTTGTCCTATGAAATT TAAATGGTAATTGATACCTCCACTAAGGAGTCAAATCAATAGCTTTGATCACAAGCAGAAGAAATGATAA GAGCGCAAAGCTTGGAATAATCTCTTTATATGTCCCATTGATTAACATCTATCCTACAAGGGCGTGAACA ACCACGATCAGAGTTTAGGAGAGCGATCAACTCCAAGCACTAAAAGGGCGTGAACAACCACTATAAGAAT TGAAGAGGCGATCCTCCAAGAACGCTGCCCGACCAGGCATTAATTGCCGCATAATCTGACCGTCGTCTGG ATAGCCTGGCAGACGGGAAAACTTCATGGAATAATGACGCCATTACTTGAAGTTTCGCGGGGCTTTTTTT CCTGGCTCGGAATTATACTTTTTACGCCATAACTGCGTTTCCCCGTTCTCCAAGGGCTCCGCCGTCGGCA ACTTTGTATGGGGAAGCGCGGTAGATTTTGTTTTCAAGGAAGGACCAAGGGTGTGAGGTGGCTTTTGAAA CACTGGTGCCATGTATGGGACTATTCTGACGGATCTCCGCCGCGCAACTTAACTCCCGCCTCGGATTCCG TGTTAGGCGTCGTAACGAACATTAGGAAGACATCGGATCTTAGATGAGAAATTATGCATCATGCGACTTG TGTGAATGTGCATCAATCTTTTGCCTGTTGCATTACGCATGCTCACGCGACTTCATGAATATGTCTTCCC TTACATACTCCTCCTGATCACGACAACACAACATTTCTCACATCACTCCAGGGTGGTCG Online Resource 2. Annotated sequence of N. gaditana NR gene. Putative CAAT boxes (bold and underlined text), TATA box (bold, underlined and highlighted blue), and possible alternative TATA box (highlighted yellow) were identified with PLACE and PlantCARE. A putative transcription start site (highlighted green) was identified with TSSPlant, although this was not confirmed experimentally.
Online Resource 5
Online Resource 5. Analysis of eGFP fluorescence in three NgLCP control strains. The normalised fluorescence measurements after subtraction of wild-type fluorescence varied between strains but were not significantly different in each strain when the nitrogen source was switched from ammonium chloride (red bars) to sodium nitrate (green bars). Values are means ± S.D. of triplicate measurements
Online Resource 6
Online Resource 6. Analysis of eGFP in ammonium and nitrate-grown NgNR3 cultures via immunoblot. NgNR3 Cultures were grown to the exponential phase in media containing either ammonium or nitrate as the sole nitrogen source before protein samples were extracted and probed with an antibody to eGFP. In the nitrate-gown culture samples and the eGFP standards, a band migrating to a distance equivalent to the expected mass of eGFP (27 kDa) was detected. In the ammonium-grown culture and the wild-type negative control, this band was not detected. Lane 1; N. gaditana wild-type (negative control), lane 2; ammonium-grown NgNR3 culture, lane 3; nitrate-grown NgNR3 culture, lane 4-8; eGFP standards (20, 40, 60, 80, 160, 320 ng)
