Gateway Vectors for BiFC
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1 Gateway Vectors for BiFC 1. The enhanced YFP (EYFP) are used (Split EYFP). 2. The Fusion fusion gene is expressed by CaMV35S promoter. 3. The N- or C-terminal fragments of EYFP are fused subsequent to the LR reaction. 4. Please read the Gateway instruction manual (Invitrogen) for procedures, etc. Please note that our BiFC vectors are available for basic research only. If you plan to use them for commercial research, please contact Invitrogen about the licencing of Gateway TM. Please do not distribute to other researchers without permission. Tsuyoshi Nakagawa Department of Molecular and Functional Genomics Center for Integrated Research in Science Shimane University Matsue , Japan
2 Construction of BiFC vectors The vector used as the starting plasmid was pugw2 and pugw0 (Nakagawa, T. et al., J. Biosci. Bioeng., 104, ) which were constructed from puc119. The EYFP (239 amino acids) were separated into neyfp (1-174) and ceyfp ( ), then introduced into pugw2 and pugw0, respectively, to make four types of vectors. (1) neyfp/pugw2 (35S pro, C-nEYFP) (2) ceyfp/pugw2 (35S pro, C-cEYFP) (3) neyfp/pugw0 (35S pro, N-nEYFP) (4) ceyfp/pugw0 (35S pro, N-cEYFP) Antibiotics All BiFC vectors : ampicillin (50 mg/l) and chloramphenicol (30 mg/l) All recombinant BiFC vectors (after LR reaction) : ampicillin (50 mg/l) Amplification of BiFC vectors (1) Use competent cell of DB3.1 (Invitrogen ) or ccdb Survival T1 (Invitrogen C ) (2) Spread on a plate containing ampicillin (50 mg/l) and chloramphenicol (30 mg/l). We made and use 10 mg/ml (methanol) stock solution of chloramphenicol. (3) Pick up colony and culture in a liquid medium containing ampicillin (50 mg/l) and chloramphenicol (30 mg/l) (4) Prepare plasmids from E. coli with plasmid purification kit. We usually use Mag Extractor plasmid (Toyobo NPK-301). LR reaction and transformation (1) Reaction mixture can be scaled down. We usually use the following reaction: (2) Entry clone 0.5 µl (about 150 ng) BiFC vector 1.0 µl (about 150 ng) LR clonase II 0.5 µl H 2 O 2.0 µl total 4.0 µl (3) Incubate at 25 ºC for more than 3hr.
3 (4) 75 ºC for 10 min. We do not use proteinase K solution to stop the reaction. (5) Add 2 µl of reaction mixture to 5 µl of DH5α competent cell. We usually use Competent High DH5α (Toyobo DNA-903). On ice for 30 min, 42 ºC for 45 sec, on ice for 2 min, add 100 µl of SOC, shake for 1hr. (6) Spread on a plate containing ampicillin (50 mg/l). Note (1) Linearization of BiFC vectors before performing the LR reaction is not necessary. If you want to linealize BiFC vectors, use XhoI. (2) Use the Escherichia coli strain DH5α for cloning the product of the LR reactions, since negative selection with ccdb does not work in the E.coli which has F episome (see the Invitrogen instruction manual). (3) The pugw2 has the structure of HindIII-XbaI-HindIII-CaMV35S pro-xbai around the CaMV35S promoter. The CaMV35S promoter is removed from pugw2 by digestion of XbaI and following self ligation. (4) BiFC vectors are used for transient expression experiments. See protocol of particle gun, protoplast transformation with PEG, etc., on the website.
4 Structure of BiFC vectors ori Amp r nyfp / pugw2 (P 35S / C-nEYFP) HindIII XbaI HindIII XhoI XbaI ccdb P 35S Cm r neyfp SacI Tnos EcoRI attr1 attr2 ori Amp r cyfp / pugw2 (P 35S / C- cyfp) HindIII XbaI HindIII XhoI XbaI ccdb P 35S Cm r ceyfp SacI Tnos EcoRI attr1 attr2 ori Amp r nyfp / pugw0 (P 35S / N-nEYFP) HindIII P 35S XbaI neyfp XhoI SacI EcoRI ccdb Cm r Tnos attr1 attr2 ori Amp r cyfp / pugw0 (P 35S / N- cyfp) HindIII P 35S XbaI ceyfp XhoI ccdb SacI EcoRI Cm r Tnos attr1 attr2 Structure of the BiFC gateway vectors. ori, ColE1 origin; Amp r, ampicillin resistant marker; Cm r, chloramphenicol resistant marker.
5 Sequence of BiFC vectors The HindIII (AAGCTT), XbaI (TCTAGA) and SacI (GAGCTC) sites are shown. The attr1-cmr-ccdb-attr2 cassette is underlined. The termination codon in the vector are bold-underlined. n/ceyfp / pugw2 (35S promoter, C-n/cEYFP) (AAGCTT)GGG(TCTAGA)CCC(AAGCTT) -- (35S promoter) --// (TCTAGA)GTTA TCA ACA AGT TTG TAC AAA AAA --(CmR,ccdB)--TTC TTG TAC AAA GTG GTT GAT AAC AGC n/ceyfp GCT TA(G AGCTC) neyfp (M1-D174) + TAG ceyfp ATG + (G175-TAA)
6 n/ceyfp / puge0 (35S promoter, N-n/cEYFP) (AAGCTT) -- 35S promoter --// (TCTAGA) ATG AGC n/ceyfp GCT GTT ATC ACA AGT TTG TAC AAA AAA -(CmR,ccdB)--TTC TTG TAC AAA GTG GTG ATA ACC TA(G AGCTC) neyfp M1-D174 ceyfp ATG + (G175-K239) K239 is last amino acid of EYFP
7 Nucleotide and amino acid sequences around attb sites after LR reaction neyfp / pugw2 (CaMV35S promoter; C-fusion to neyfp)!!"#$$"""$#$!"!###!!"#$$"#!$"##$"#!""$%&#'()*+,-%#"#!!"!###$$##$#$!$!$!!""!!"$$#!$$$#!$$$""!"!"!!#!#"""""!#$#$!"!"$$!! $#!!!#!!!"$!$$"!$!#!!!!!!!!!"#!!""#!$..!&/0123-!.!#!##!!"#$!$$#!$$"!$!#!!!!!"$"!"$$!"!$!!!#!!"#!!$"%&24567-%!"!#!$!"!"#$!$!"!!"#$#!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 8!!!7!!!!!!!6!!!9!!!5!!!:!!!)!!!)!!!;!!!.!!!,!!!(!!!!!!!!!!!!;!!!!!!!!!!!!!!!!!!!!'<<=>!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!'<<=?!!!!! ceyfp / pugw2 (CaMV35S promoter; C-fusion to ceyfp)!!"#$$"""$#$!"!###!!"#$$"#!$"##$"#!""$%&#'()*+,-%#"#!!"!###$$##$#$!$!$!!""!!"$$#!$$$#!$$$""!"!"!!#!#"""""!#$#$!"!"$$!! $#!!!#!!!"$!$$"!$!#!!!!!!!!!"#!!""#!$..!&/0123-!.!#!##!!"#$!$$#!$$"!$!#!!!!!"$"!"$$!"!$!!!#!!"#!!$"%""#%&/4567-%!!!"%$!!!"#$!$!"!!"#$#!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 8!!!7!!!!!!!6!!!9!!!5!!!:!!!)!!!)!!!;!!!.!!!,!!!(!!!"!!!!!!!!!!!!:!!!!!!!!!!!!!!!!!!!!!'<<=>!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!'<<=?!!!!! nefp / pugw0 (CaMV35S promoter; N-fusion to neyfp)!!"#$$"#!$"##$"#!""$%&#'()*+,-%#"#!!"!###$$##$#$!$!$!!""!!"$$#!$$$#!$$$""!"!"!!#!#"""""!#$#$!"!!$"!!"#!!$"%&24567-%"!#!"#$!"$$!!$#!!#!!!"$!$$"!$!#!!!!!!!!!"#!!""#!$..!&/0123-!.!#!##!!"#$!$$#!$$"!$!#!!!!!"$"!"$"!!$!!!##!$!"!!"#$# (!!!,!!!(!!!!!!!!!!!;!!!!!!!)!!!@!!!$!!!,!!!9!!!5!!!:!!!:!!!!!!!"!!!8!!!!!!!!!!!8!!!7!!!!!!!6!!!9!!!5!!!:!!!)!!!)!!!@!!!$!!!A!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '<<=>!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!'<<=?!!!! nefp / pugw0 (CaMV35S promoter; N-fusion to ceyfp)!!"#$$"#!$"##$"#!""$%&#'()*+,-%#"#!!"!###$$##$#$!$!$!!""!!"$$#!$$$#!$$$""!"!"!!#!#"""""!#$#$!"!!$"!!"#!!$"%""#%&/4567-%!!"!"#$!"$$!!$#!!#!!!"$!$$"!$!#!!!!!!!!!"#!!""#!$..!&/0123-!.!#!##!!"#$!$$#!$$"!$!#!!!!!"$"!"$"!!$!!!##!$!"!!"#$# (!!!,!!!(!!!"!!!!!!!!!!!:!!!!!!!)!!!@!!!$!!!,!!!9!!!5!!!:!!!:!!!!!!!"!!!8!!!!!!!!!!!8!!!7!!!!!!!6!!!9!!!5!!!:!!!)!!!)!!!@!!!$!!!A!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '<<=>!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!'<<=?!!!!
8 The sequences between HindIII and Sac I sites (see Structure of BiFC vectors ) are shown for all vectors. To clearly show the relation of functional elements, linefeeds are irregularly inserted in the sequences. The attb1 and attb2 sites are underlined. N indicates a nucleotide derived from an entry clone (green) and X indicates an amino acid residue corresponding to the N-containing codon. The CaMV35S promoter is shown in orange, where the region between -46 and +8 is represented by a nucleotide sequence with the predicted transcription start site (underlined larger letter). The neyfp is indicated in red and cyfp is indicated in blue. Only partial sequences are shown for n/ceyfp, whose whole sequences are indicated in the former section. In C-fusion vectors, the peptide sequence derived from attb2 region becomes the linker between the entry clone and n/ceyfp. In N-fusion vectors, the peptide sequence derived from attb1 region becomes the linker between n/ceyfp and the entry clone, and the peptide sequence derived from attb2 region (gray italic) is added at the C-terminus of the fusion protein only if the entry clone lacks the termination codon in it.
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