Tzfira et al.
pSAT vectors: a modular series of plasmids for autofluorescent protein tagging and
expression of multiple genes in plants
Tzvi Tzfira1*, Guo-Wei Tian1, Benoît Lacroix1, Shachi Vyas1, Jianxiong Li1, Yael LeitnerDagan2, Alexander Krichevsky1, Tamir Taylor3, Alexander Vainstein2, and Vitaly Citovsky1
1
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY
11794-5215. 2The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture,
Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of
Jerusalem, Rehovot 76100, Israel. 3Ward Melville High School, 380 Old Town Road, East
Setauket, NY 11733
Running title: pSAT vectors for autofluorescent protein tagging and multiple gene expression
Key words: autofluorescent proteins, plasmids, multiple gene expression
*
Corresponding Author:
Phone: 631-632-1004
Fax: 631-632-8575
E-mail: ttzfira@ms.cc.sunysb.edu
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Supplement 1. A complete list of plasmids described in this paper and available for distribution.
The plasmids listed in Tables 1a and 1b will be distributed by the State University of New York
(SUNY). Requests should include the full name of the plasmid as it appears in the Tables below and
should be sent by electronic mail to the corresponding author (TT) at ttzfira@ms.cc.sunysb.edu.
Table 1a. List of basic pSAT plasmids and Gateway™ destination pSAT plasmids
Plasmid name
AFP tag
Restriction sites flanking
the expression cassette
MCS location
GenBank accession
number
pSAT6-EGFP-C1
pSAT6-EYFP-C1
pSAT6-ECFP-C1
pSAT6-DsRed2-C1
pSAT6-Citrine-C1
EGFP
EYFP
ECFP
DsRed2
Citrine-YFP
PI-PspI
PI-PspI
PI-PspI
PI-PspI
PI-PspI
C1
C1
C1
C1
C1
AY818377
AY818380
AY818374
AY818375
AY819771
pSAT6-EGFP-N1
pSAT6-EYFP-N1
pSAT6-ECFP-N1
pSAT6-DsRed2-N1
pSAT6-Citrine-N1
EGFP
EYFP
ECFP
DsRed2
Citrine
PI-PspI
PI-PspI
PI-PspI
PI-PspI
PI-PspI
N1
N1
N1
N1
N1
AY818382
AY818378
AY818381
AY818373
AY818369
EGFP
EGFP
EGFP
EGFP
EGFP
EGFP
AscI
I-PpoI/AscI
I-PpoI
PI-SceI
PI-CeuI
PI-TliI
C1
C1
C1
C1
C1
C1
AY818363
AY818365
AY818366
AY818367
AY818368
AY818384
N/A
PI-PspI
N/A
AY818383
EGFP
EGFP
EGFP
PI-PspI
PI-PspI
PI-PspI
C1a
N1a
Instead of
promotera
AY818372
AY818370
AY818379
Basic pSAT plasmids
pSAT1-EGFP-C1
pSAT2-EGFP-C1
pSAT3-EGFP-C1
pSAT4-EGFP-C1
pSAT5-EGFP-C1
pSAT7-EGFP-C1
pSAT6-MCS
Gateway™ pSAT plasmids
pSAT6-DEST-EGFP-C1
pSAT6-DEST-EGFP-N1
pSAT6-NP-DEST-EGFP
a
Location of the Gateway™ destination cassette.
Table 1b. List of selectable pSAT and pRSC2 plasmids
Plasmid name
pSAT1-hpt
pSAT4-nptII
pSAT6-bar
pRCS2-hpt
pRCS2-nptII
pRCS2-bar
Selection marker
Hygromycin
Kanamycin
Phosphinothricin
Hygromycin
Kanamycin
Phosphinothricin
GenBank accession number
AY818364
AY818371
AY818376
Unavailable
Unavailable
Unavailable
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Supplement 2. A detailed description of plasmid construction methods.
Construction of pSAT vectors
The original MCS of pUC18 (Norrander et al., 1983) was replaced by PCR amplification of the
entire plasmid backbone using the primers
5’AAATACTGCAGCCATGGAATTCTAGAGCGGCCGCGTAATCATGGTCATAGCTGTTT
CC3’ and
5’AAATACTGCAGGTCGACGAATTCACCGGTGGCACTGGCCGTCGTTTTACAACG3’
which carried the sequences for the new MCS. The PCR product was digested with PstI and selfligated, resulting in pUC18-MCS-A which contained the following restriction endonuclease
recognition sites in its MCS: AgeI, EcoRI, SalI, PstI, NcoI, EcoRI, XbaI, and NotI. To construct a
plant gene expression cassette, the tandem CaMV 35S promoter was PCR-amplified from
pRTL2-GUS (Restrepo et al., 1990) and inserted into the AgeI-SalI sites of pUC18-MCS-A,
producing pUC18-2x35Sp. Next, the TEV leader (TL) sequence was PCR-amplified from
pRTL2-GUS and cloned as a XhoI-NcoI fragment into the SalI-NcoI sites of pUC18-2x35Sp,
producing pUC18-2x35Sp-TL. A single A=>G mutation was introduced in one of the TL
primers to eliminate an internal EcoRI site located at the 5’end of the TL fragment. Finally, the
EGFP ORF with its adjacent MCS and the CaMV 35S terminator were PCR-amplified from
pEGFP-C1 (Clontech, San Jose, CA) and pRTL2-GUS, respectively, and cloned by triple
ligation as NcoI-XbaI and XbaI-NotI fragments into the NcoI-NotI sites of pUC18-2x35Sp-TL.
The resulting EGFP-C1 plant expression cassette was then transferred into the AgeI-NotI sites of
pAUX3133 (Goderis et al., 2002), producing pSAT6-EGFP-C1.
To produce pSAT6-EYFP-C1 and pSAT6-ECFP-C1, EYFP-C1 and ECFP-C1 ORFs and
their adjacent MCSs were PCR-amplified from pEYFP-C1 and pECFP-C1 (Clontech, San Jose,
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CA), respectively, and cloned into the NcoI-XbaI sites of pSAT6-EGFP-C1, replacing EGFP-C1MCS. To produce pSAT6-Citrine-C1, the Citrine-YFP ORF was PCR-amplified from pRSETBCitrine (Griesbeck et al., 2001) and cloned into the NcoI-XhoI sites of pSAT6-EGFP-C1,
replacing EGFP. The Citrine-YFP PCR fragment was designed to contain BspEI and BglII
recognition sites to reconstruct the complete MCS originally present in pSAT6-EGFP-C1.
For pSAT6-MCS, the entire MCS and its adjacent 35S terminator was PCR-amplified
from pSAT6-EYFP-C1, cloned into the NcoI-NotI sites of pUC18-2x35Sp-TL, and, then, the
complete expression cassette was transferred into the AgeI-NotI sites of pAUX3133.
To produce pSAT6-EGFP-N1, pSAT6-EYFP-N1, pSAT6-ECFP-N1 and pSAT6-Citrine,
the EGFP, EYFP, ECFP and Citrine ORFs were PCR-amplified as SalI-BglII fragments—
designed to reconstruct all MCS sites originally present in pSAT6-MCS and to allow in-frame
fusions to the N-terminus of each AFP—from pEGFP-C1, pEYFP-C1, pECFP-C1 and pRESTB
respectively, and cloned into the SalI-BamHI sites of pSAT6-MCS.
To produce pSAT6-DsRed2-C1, the DsRed2 ORF was cloned by triple-ligation of NcoI
and NcoI-XhoI fragments of pDsRed2-C1 (Clontech, San Jose, CA) into the NcoI-XhoI sites of
pSAT6-EGFP-C1, instead of GFP. To produce pSAT6-DsRed2-N1, the DsRed2 ORF was PCRamplified as a SalI-BglII fragment—designed to reconstruct all MCS sites originally present in
pSAT6-MCS and to allow in-frame fusions to the N-terminus of DsRed2—from pDsRed2-C1
and cloned into SalI-BamHI sites of pSAT6-MCS.
All PCR reactions were performed using a high fidelity Pfu DNA polymerase
(Stratagene), and their products were verified by DNA sequencing. Full DNA sequences of all
pSAT6 vectors were deposited to the GenBank and assigned the following accession numbers:
AY818377, AY818380, AY818374, AY819771, AY818375, AY818382, AY818378,
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AY818381, AY818369, AY818373 and AY818383 for pSAT6-EGFP-C1, pSAT6-EYFP-C1,
pSAT6-ECFP-C1, pSAT6-Citrine-C1, pSAT6-DsRed2-C1, pSAT6-EGFP-N1, pSAT6-EYFPN1, pSAT6-ECFP-N1, pSAT6-Citrine-N1, pSAT6-DsRed2-N1, and pSAT6-MCS, respectively.
For pSAT1, pSAT2, pSAT3, pSAT4, pSAT5, and pSAT7 versions of pSAT6-EGFP-C1,
the GFP expression cassette was transferred into the AgeI-NotI sites of pAUX3166, pAUX3130,
pAUX3169, pAUX3131, pAUX3132, and pAUX3167 (Goderis et al., 2002), producing pSAT1EGFP-C1, pSAT2-EGFP-C1, pSAT3-EGFP-C1, pSAT4-EGFP-C1, pSAT5-EGFP-C1 and
pSAT7-EGFP-C1, respectively, with the corresponding GenBank accession numbers AY818363,
AY818365, AY818366, AY818367, AY818368, and AY818384.
Cloning of tested genes into pSAT vectors
The tested genes were cloned into the MCSs of pSAT vectors. Specifically, the Arabidopsis
VIP1 was inserted as a SalI-BamHI fragment from pRTL2-GFP-VIP1 (Avivi et al., 2004) into
pSAT6-EGFP-C1, producing pSAT6-EGFP-C1-VIP1. The Arabidopsis CHS
(PelletierandShirley, 1996) was cloned as a BamHI-KpnI fragment from pCHS.CR2 (kindly
provided by Dr. Shirley, Virginia Polytechnic Institute and State University) into the BglII-KpnI
sites of pSAT6-EYFP-C1, producing pSAT6-EYFP-C1-CHS. The full-length, unprocessed ORF
of cucumber chrC was PCR-amplified from pBS-KS-cucChrC (Vishnevetsky et al., 1996) and
cloned into the SalI-BamHI sites of pSAT6-EGFP-N1, producing pSAT6-EGFP-N1-cucChrC.
The Arabidopsis TUA2 ORF was PCR-amplified from pU12436 (Arabidopsis Biological
Resource Center, ARBC) and cloned into the EcoRI-BamHI sites of pSAT6-DsRed2-C1,
producing pSAT6-DsRed2-C1-TUA2.
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Assembly of several pSAT expression cassettes into single plasmids
For expression of two autofluorescently-tagged proteins from a single plasmid, we first produced
pSAT1-ECFP-C1-VIP1 by cloning the VIP1 ORF as a SalI-BamHI fragment from pSAT6EGFP-C1-VIP1 into pSAT1-ECFP-C1. The ECFP-VIP1 expression cassette was then cloned as
an AscI fragment of pSAT1-ECFP-C1-VIP1 into pPZP-RCS2, producing pPZP-ECFP-VIP1.
Next, the EYFP-CHS expression cassette was inserted into pPZP-ECFP-VIP1 as a PI-PspI
fragment from pSAT6-EYFP-C1-CHS, producing pPZP-ECFP-VIP1/EYFP-CHS. For
expression of three autofluorescently-tagged proteins from a single plasmid, we first transferred
EYFP-CHS as a NcoI-BamHI fragment from pSAT6-EYFP-C1-CHS into pSAT1-EYFP-C1 to
produce pSAT1-EYFP-C1-CHS. Next, the SYNV P protein ORF (Goodin et al., 2001; Goodin et
al., 2002) was cloned as a SalI-BamHI fragment into pSAT4-DsRed2-C1, resulting in pSAT4DsRed2-C1-P. Finally, the ECFP, EYFP-CHS and DsRed2-P expression cassettes were cloned
as PI-PspI, AscI and I-SceI fragments from pSAT6-ECFP-C1, pSAT1-EYFP-C1-CHS and
pSAT4-DsRed-C1-P into pPZP-RCS2, producing pRCS2-EYFP-CHS/DsRed2-P/ECFP. In
addition, the SYNV N protein ORF was cloned as a SalI-BamHI fragment into pSAT3-MCS,
producing pSAT3-N.
Promoter and terminator replacement in pSAT vectors
The nopaline synthase promoter (nosP) was PCR-amplified from pBI101 (Clontech) and cloned
into the AgeI-NcoI PCR sites of pSAT6-EGFP-C1, replacing 35S promoter and TL sequences,
producing pSAT6-nosP-EGFP-C1. The nopaline synthase terminator (nosT) PCR-amplified
from pBI101 was then cloned into the XbaI-NotI sites of pSAT6-nosP-EGFP-C1, replacing the
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35S terminator and producing pSAT6-nosPnosT-EGFP-C1. A DNA fragment containing three
copies of the Gal4 UAS and the TATA box from the 35S promoter were cloned by triple ligation
of AgeI-EcoRI and MfeI-NcoI PCR fragments, respectively, from pTA7001 (AoyamaandChua,
1997) into the AgeI-NcoI sites of pSAT6-MCS, replacing 35S promoter and its adjacent TL
sequence and producing pSAT6-uasP-MCS. The uidA gene encoding GUS was then cloned from
pRTL2-GUS into the NcoI-XbaI sites of pSAT6-uasP-MCS, producing pSAT6-uasP-GUS. The
mGAL4-VP16 activator was cloned as a BspHI-XbaI PCR fragment from pET-15GAL4VP16UASmGFP5ER (kindly provided by Dr. Haseloff, University of Cambridge, UK) into NcoI
and XbaI sites of pSAT6-MCS, producing pSAT6-mGAL4VP16.
A 140-bp portion of the chrC promoter (140P, A.V., unpublished) contained within an
AgeI-NcoI fragment was used to replace the 35S promoter and TL sequences in pSAT1-EYFPC1, resulting in pSAT1-140P-EYFP-C1. The VIP1 ORF was then transferred from pSAT6EGFP-C1 into the SalI-BamHI sites of pSAT1-140P-EYFP-C1, producing pSAT1-140P-EYFPC1-VIP1. The expression cassettes from pSAT1-140P-EYFP-C1-VIP1 and pSAT6-DsRed2-C1
were cloned into pPZP-RCS2 as AscI and PI-PspI fragments, respectively, producing pRCS2140P-EYFP-VIP1/DsRed2. The mybys ORF (A.V., unpublished) was PCR-amplified and cloned
into the XhoI-BamHI sites of pSAT1-MCS, producing pSAT1-mybys. The expression cassettes
from pSAT1-mybys and pSAT6-ECFP-C1 were cloned into pPZP-RCS2 as AscI and PI-PspI
fragments, respectively, producing pRCS2-mybys/ECFP.
Construction of pRCS2 binary plasmids with selectable markers for transgenic expression
Selectable markers were first cloned into pSAT vectors and then transferred to pPZP-RCS2.
Specifically, the hpt gene was PCR-amplified from pBIG-HYG (Tzfira et al., 1997) and cloned
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into the HindIII-BamHI sites of pSAT6-MCS, producing pSAT1-hpt. The nptII gene was PCRamplified from pBI101 and cloned into the SalI-BamHI sites of pSAT4-MCS, producing pSAT4nptII. The bar gene was PCR-amplified from pFGC5941 (ARBC) and cloned into the XhoI-XmaI
sites of pSAT6-MCS, producing pSAT1-bar. From these pSAT constructs, the resistance gene
expression cassettes were transferred as AscI, I-SceI, and PI-PI-PspI fragments into the
corresponding sites of pPZP-RCS2, resulting in RCS2-hpt, RCS2-nptII, and RCS2-bar,
respectively. Finally, we cloned the expression cassettes from pSAT1-EGFP-C1 and pSAT6DsRed2-C1 into the AscI and PI-PspI sites, respectively, of pRCS2-nptII to produce pRCS2nptII-DsRed2/EGFP. Complete DNA sequences of pSAT1-hpt, pSAT4-nptII, and pSAT6-bar
were deposited to the GenBank and assigned accession numbers AY818364, AY818371 and
AY818376, respectively.
Conversion of pSAT6 to Gateway destination vectors
The Gateway conversion cassette (reading frame A, Invitrogen) was PCR-amplified using the
forward 5’GGGGGAGATCTGGATCACAAGTTTGTACAAAAAAGC3’ and reverse
5’GGGGGGGGCCCTATCACCACTTTGTACCAG3’ primers and cloned into the BglII-ApaI
sites of pSAT6-EGFP-N1, replacing MCS and producing pSAT6-DEST-EGFP-N1. The same
conversion cassette was also PCR-amplified using the forward
5’GGGGGAGATCTATCACAAGTTTGTACAAAAAAGC3’ and reverse
5’GGGGGTCTAGATATCACCACTTTGTACAAG3’ primers and cloned into the BglII-XbaI
sites of pSAT6-EGFP-C1, replacing MCS and producing pSAT6-DEST-EGFP-C1. Finally, the
same conversion cassette was PCR-amplified using the forward
5’GGGGGACCGGTATCACAAGTTTGTACAAAAAAGC3’ and reverse
5’GGGGGTCATGACTATCACCACTTTGTACAAG3’ primers and cloned into the AgeI-BspHI
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sites of pSAT6-EGFP-C1, replacing the 35S promoter and TL sequences and producing pSAT6NP-DEST-EGFP-C1. Plasmids were maintained in E. coli DB3.1 strain due to toxicity of the
ccdB gene contained within the conversion cassette in most other bacterial strains. Complete
DNA sequences of pSAT6-DEST-EGFP-N1, pSAT6-DEST-EGFP-C1, and pSAT6-NP-DESTEGFP-C1 were deposited to the GenBank and assigned accession numbers AY818370,
AY818372 and AY818379, respectively.
For production of pDONR-nosP, nosP was first PCR-amplified from pBI101t using the
forward 5’AAAAAGCAGGCTTCGATCATGAGCGGAGAATTAAG3’ and reverse
5’AGAAAGCTGGGTCGGATTGAGAGTGAATATGAGAC3’ primers. The resulting fragment
was re-amplified using the Gateway universal forward
(5’GGGGACAAGTTTGTACAAAAAAGCAGGCT3’) and reverse
(5’GGGGACCACTTTGTACAAGAAAGCTGGGT3’) primers, that generate the attB1 and atB2
sites flanking the fragment (Walhout et al., 2000), and recombined by the BP clonase reaction
into the attP1 and attP2 sites of the donor vector pDONR207 (Invitrogen) as described (Tian et
al., 2004); this reaction produces the attL1 and attL2 sequences that flank the cloned gene within
the donor vector (Walhout et al., 2000). From pDONR-nosP, nosP was recombined into the
attR1 and attR2 sites of the destination vector pSAT6-NP-DEST-EGFP-C1 by the LR clonase
reaction (Tian et al., 2004), resulting in pSAT6-nosP-DEST-EGFP.
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