2in1 Plasmid Toolkit
(Kit # 1000000147, 1000000148, 1000000149, 1000000150 )

Depositing Lab:   Christopher Grefen

The Grefen lab has merged a 2in1 vector cloning system with Förster Resonance Energy Transfer (FRET) or ratiometric Bimolecular Fluorescence Complementation (rBiFC, Xing et al. 2016) to allow the improved analysis of protein-protein interaction in planta.

These kits comprise 4 different, binary 2in1 vectors with all possible combinations of tag orientation (NN, NC, CN and CC).

The kits will be sent as individual bacterial stabs at room temperature. Individual plasmids can be ordered from each plasmid page and will be shipped as bacterial stabs.

Description

The 2in1 destination vectors allow the analysis of protein-protein interaction in planta via Förster Resonance Energy Transfer (FRET) or ratiometric Bimolecular Fluorescence Complementation (rBiFC, Xing et al. 2016).

2in1 cloning reactions require dedicated Entry clones. These can either be created using commercially available Entry vectors pDONR221-P1P4™ or pDONR221-P3P2™ (Thermofisher) via BP reaction or using our own Entry vectors pUC-L1L4 and pUC-L3L2.

The pUC vectors have the advantage that one can use classical cloning methods to introduce the gene of interest (GOI). PCR-amplify your GOI adding either an AvrII/NheI/SpeI or XbaI 5’ of and in frame with the ATG and a blunt-end enzyme of your choice at the 3’ end (in frame if C-terminal fusions are planned or with a stop codon if not). The Chloramphenicol/ccdB cassette in the pUC vectors can be excised using XbaI/NaeI [L1L4] or NheI/NaeI [L3L2], respectively. Insert your GOI via standard ligation procedure. Use ccdB sensitive bacteria such as Top10 or XL1blue to reduce false positives.

After sequence verification of your Entry clones, perform a 2in1-cloning reaction using LR clonase (Thermofisher) and as described in Grefen and Blatt 2012 and Mehlhorn et al. 2018.

The kits comprise 4 different, binary 2in1 vectors with all possible combinations of tag orientation (NN, NC, CN and CC). Kit #1000000147 contains nYFP/cYFP fluorophores plus soluble RFP for rBiFC ( Grefen and Blatt 2012). For FRET analyses, kit #1000000148 contains eGFP/mCherry, kit #1000000149 contains mTRQ2/mVenus, and kit #1000000150 contains mVenus/tagRFP fluorophores ( Hecker et al. 2015). All 4 kits also contain the two Entry vectors pUC-L1L4 and pUC-L3L2.

General Resources for 2in1 Cloning and Protein-Protein Interaction in planta

Techniques for the analysis of protein-protein interactions in vivo. Xing S, et al. Plant Physiology, 2016, Jun;171(2):727-58. PubMed PMID 27208310

2in1 Vectors Improve In Planta BiFC and FRET Analyses. Mehlhorn DG, et al. Methods Mol Biol., 2018, 1691:139-158. PubMed PMID 29043675

Click below to download more information about the 2in1 cloning principle:

2in1 Cloning Principle 76.1 KB

Kit-Specific Resources

rBiFC kit: A 2in1 cloning system enables ratiometric Bimolecular Fluorescence Complementation (rBiFC). Grefen C and Blatt MR. Biotechniques, 2012, Oct 2;53(5):311-14. PubMed PMID 23066669

FRET kits: Binary 2in1 Vectors Improve in Planta (Co)localization and Dynamic Protein Interaction Studies. Hecker A, et al. Plant Physiology, 2015, Jul;168(3):776-87. PubMed PMID 25971551

How to Cite this Kit

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.

For your Materials and Methods section:

“The 2in1 Plasmid Toolkit was a gift from Christopher Grefen (Addgene kit #10000001XX).”

For your Reference section:

2in1 Vectors Improve In Planta BiFC and FRET Analyses. Mehlhorn DG, et al. Methods Mol Biol., 2018, 1691:139-158. PubMed PMID 29043675

2in1 plasmids improve coexpression ratios compared to classical mixing of two individually transformed plasmids (here measured through mCherry/GFP ratios of transformed tobacco nuclei).

Ratiometric Bimolecular Fluorescence Complementation (rBiFC) Kit - #1000000147

This kit contains 6 plasmids. Please refer to the individual plasmid pages below for more details on each plasmid in this kit.

Addgene ID	Plasmid Name
114018	pUC-L1L4
114019	pUC-L3L2
105111	pBiFCt-2in1-NN
105112	pBiFCt-2in1-NC
105113	pBiFCt-2in1-CN
105114	pBiFCt-2in1-CC

The signal of complemented YFP can be ratioed against the cytosolic RFP. The latter serves as both transformation control and ratiometric marker allowing to distinguish true from false positives.

EGFP/mCherry FLIM/FRET Kit - #1000000148

This kit contains 6 plasmids. Please refer to the individual plasmid pages below for more details on each plasmid in this kit.

Addgene ID	Plasmid Name
114018	pUC-L1L4
114019	pUC-L3L2
105124	pFRETgc-2in1-NN
105121	pFRETgc-2in1-NC
105118	pFRETgc-2in1-CN
105115	pFRETgc-2in1-CC

Normalized absorbance (dashed lines), emission spectra (solid lines), and λ⁴-weighted spectral overlap (light gray surface plot) for the fluorophore pair in this kit.

pmTRQ2/mVenus FLIM/FRET Kit - #1000000149

This kit contains 6 plasmids. Please refer to the individual plasmid pages below for more details on each plasmid in this kit.

Addgene ID	Plasmid Name
114018	pUC-L1L4
114019	pUC-L3L2
105125	pFRETtv-2in1-NN
105122	pFRETtv-2in1-NC
105119	pFRETtv-2in1-CN
105116	pFRETtv-2in1-CC

Normalized absorbance (dashed lines), emission spectra (solid lines), and λ⁴-weighted spectral overlap (light gray surface plot) for the fluorophore pair in this kit.

mVenus/tagRFP FLIM/FRET Kit - #1000000150

This kit contains 6 plasmids. Please refer to the individual plasmid pages below for more details on each plasmid in this kit.

Addgene ID	Plasmid Name
114018	pUC-L1L4
114019	pUC-L3L2
105126	pFRETvr-2in1-NN
105123	pFRETvr-2in1-NC
105120	pFRETvr-2in1-CN
105117	pFRETvr-2in1-CC

Normalized absorbance (dashed lines), emission spectra (solid lines), and λ⁴-weighted spectral overlap (light gray surface plot) for the fluorophore pair in this kit.