Mammalian “Fold ‘N’ Glow” Split GFP S11 Plasmid

INTENDED USE: FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES

 Description:

pCMV-mGFP Cterm S11 Neo Kan vector encodes a mammalian-codon optimized version of the 16 amino-acid engineered split GFP S11 detector fragment (amino acids 215 to 230 of the 238 amino acid GFP) for tagging proteins with a C-terminal strand 11 of GFP “GFP S11”. These GFP S11-tagged proteins can be detected using the GFP 1-10 “detector” fragment (GFP amino acids 1 to 214), a highly engineered variant of strands 1-10 of green fluorescent protein (GFP 1-10 D7 optimum). Engineering and characteristics of the bacterial codon optimized version of this optimized split GFP system was originally described in (1). GFP S11 contains 3 amino acid substitutions and truncation of the last 8 amino acids of GFP (listed on page 2), which improve the solubility of the protein and increase the rate of fluorescence formation when complemented with GFP 1-10. Using the in vivo bacterial optimized two-plasmid system described in (1) to express GFP S11-tagged proteins, GFP fluorescence is easily detectable within 15 minutes after induction of the GFP 1-10 detector strand in E. coli cells. Transfer of these bacterial codon sequences to mammalian expression vectors gives detectable fluorescence, but can take up to 24 h to appear after co-transfection of the GFP 1-10 and GFP S11 constructs (2). To overcome this limitation yet retain the improved folding properties of the GFP S11 (1), the amino acid sequence of the GFP S11 expressed by pCMV-mGFP Cterm S11 Neo Kan is the same as the one described in (1), but the coding sequence is mouse codon-optimized for high expression in mammalian cells. Consequently, the fluorescence of mammalian cells co-
transfected with test proteins cloned into the MCS of pCMV-mGFP Cterm S11 Neo Kan and co-transfected with pCMV- mGFP 1-10 Hyg Amp (which contains the mammalian codon optimized GFP 1-10, available from Sandia Biotech) are up to 50-fold brighter than mammalian cells expressing the bacterial codon versions of the split GFP fragments (see Fig. 2 below). When GFP 1-10 mammalian-optimized is expressed in mammalian cell cultures co-expressing GFP S11, green- emitting cells can be detected by either fluorescence microscopy or flow cytometry within 6 to 8 h after transfection. GFP from complemented GFP 1-10 + GFP S11 has excitation and emission maxima = 488 nm and 525 nm, respectively). Complemented GFP 1-10 + GFP S11 is stable and most likely forms the same structure as full-length GFP. The mGFP S11 gene is positioned downstream of a multiple cloning site and linker just after the immediate early promoter of cytomegalovirus (P CMV IE ). As a result, cells transfected with this vector will constitutively express the proteins of interest as POI-linker-GFP S11. SV40 polyadenylation signals downstream of the GFP S11 gene direct proper processing of the 3' end of the POI-linker-GFP S11 mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded r DNA production. A neomycin-resistance cassette (Neo ) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and
polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter upstream of the cassette expresses kanamycin resistance in E. coli. 

Use
pCMV-mGFP Cterm S11 Neo Kan vector is designed as: (1) a source of the mammalian-codon optimized GFP S11 coding sequence and (2) to produce proteins of interest with C-terminal fused GFP S11 tagging peptide in mammalian cells. These can be detected by GFP 1-10 either in vivo by co-transformation with pCMV-mGFP 1-10 Hyg Amp (expressing the (available as a ready-to-use solution from Sandia Biotech). GFP fluorescence can be detected by fluorescence microscopy, providing direct visual evidence of complementation of GFP 1-10 and POI-linker-GFP S11 (see Fig. 2). After cotransfection with pCMV-mGFP 1-10 Hyg Amp vector along with a construct expressing a GFP S11-tagged protein of interest, cells can also be sorted by flow cytometry (FACS) to enrich for transfected cells, or observed by microscopy to monitor GFP 1-10 and GFP S11-tagged protein expression, interaction, translocation, or to label structures and organelles. pCMV-mGFP Cterm S11 Neo Kan vector can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (4). mammalian codon optimized GFP 1-10, available from Sandia Biotech), or by adding the GFP 1-10 detector reagent protein.

Figure 1: Using the split GFP mammalian system to follow nuclear localization of MBD2. HEK cells were co-transfected with pCMV MBD2-GFP Cterm S11 Kan Neo and pCMV-mGFP 1-10 Hyg Amp. GFP S11 is the small 16 amino acid strand 11 peptide of GFP that is detected by the GFP 1-10 by complementation to form fluorescent 11-stranded GFP. MBD2 is known to translocate to nuclei (Fig 2A). GFP 1-10 complements the GFP S11 tag, and the resulting GFP fluorescence is translocated to the nuclei (Fig 2B). Since MBD2 is expressed with only the short GFP S11 tag, and subsequently complemented with GFP 1-10, there is minimal folding perturbation compared to expressing MBD2-GFP as a direct full-length GFP fusion. Nuclear fluorescence is bright and non-punctate as expected.

REAGENTS:

Components Supplied:
Mammalian optimized split-GFP Strand 11 Plasmid: 100ng store at -20^oC.
Reagent Storage: Store all reagents between 2 o and -20 o C as listed on each kit component. When stored properly, the reagents are stable until the date indicated either on the box or each component. Depending on the particular usage requirements, it may be appropriate to re-aliquot reagents to smaller working volumes to avoid repeated freeze-thawing or repeated pipetting from the same vial. 

Materials required, but not supplied:

• Competent E.coli
• Kanamycin
• LB growth media and plates
• Restriction enzymes
• Ligation materials
• Plasmid Isolation Reagents
• Neomycin
• Fluorescence readers for detection

A . Plasmid Vector Propagation and Construction of Custom Fusion Protein Vector

1. To ensure that you have a renewable source of plasmid DNA, transform each of the plasmid vectors provided in this kit in an E.coli host strain. It is suggested to use ≥20µl of supplied plasmid for a standard chemically competent E.coli bacterial transformation.
• It is recommended that bacterial frozen stocks be prepared of all transformed plasmids using standard molecular biology techniques.
2. Purify plasmid DNA for cloning using Plasmid Preparation kits or other techniques (not included).
• It is recommended that all selected transformed plasmids under go verification testing such as by
3. restriction enzyme digest prior to sub-cloning of your gene of interest (GOI).
4. The Mammalian optimized strand-11 plasmid features a multiple cloning site (MCS) that offers several unique, conveniently arranged restriction sites for insertion of the transcription factor activation domain sequence. Expression of the fusion protein driven by the CMV promoter, a strong promoter that allows high-level constitutive expression in a variety of cell lines and the neomycin-resistance gene, which facilitates selection of stable cell lines that express the fusion protein of interest.

B. Cloning:

Notes: The pCMV-mGFP s11 vector contains a stop codon at the end of the reporter gene so they are most suitable for cloning your gene of interest upstream of the reporter gene (i.e. C-terminal fusion). The inserted gene (POI or GOI) should include the initiating ATG codon. Make sure that your gene of interest does not contain a stop codon at its end if you desire to obtain a fusion with the reporter gene downstream.

1. Prepare protein gene of interest by adding restriction enzyme sites on both ends of GOI fragment; this can be done by PCR or by digesting GOI fragment out of a plasmid with restriction enzyme sites compatible with the pCMV- mGFP s11 fusion vector.
2. Digest the pCMV-mGFP s11 fusion vector with the chosen restriction enzyme(s). Make sure that enzyme sites are not too close to each other to avoid digestion interference, and that enzyme sites are not blocked by methylation.
3. Ligate the GOI fragment into the linearized pCMV-mGFP s11 fusion vector using the ligation enzyme supplier’s recommendations.

C. Transformation:

1. To ensure that you have a renewable source of your customized fusion protein, transform the plasmid in a E.coli host strain.
• It is recommended that bacterial frozen stocks be prepared of all transformed plasmids using standard molecular biology techniques.
2. Purify plasmid DNA for mammalian transfection using Plasmid Preparation kits or other techniques (not included).
• It is recommended that all selected transformed plasmids under go verification testing such as by restriction enzyme digest prior to sub-cloning of your gene of interest (GOI).

D. Transfection:

Plasmid DNA for transfection into mammalian cells must be clean and free of phenol and sodium chloride. Transfection methods include calcium phosphate, cationic lipids, and electroporation techniques (not included). The pCMV-mGFP s11 vector is designed to be co-transfected with pCMV-mGFP s1-10 vector or used with GFP 1-10 Fold n’ Glow detector solution for detection of the fusion protein by florescence.

SEQUENCE INFORMATION

• Detailed sequence information is available on request.

pCMV-mGFP Cterm S11 Neo Kan Vector Information: Catalog Number 22004003

MCS (613-681)* BglII (610) XhoI (614) SacI (621) HIndIII (623) EcoRI (630) PstI (639) SalI (640) AccI (641) KpnI (650) SacII (653) XmaI (657) SmaI (659) AgeI (667) ApaI (658)

Figure 2. Feature Map of pCMV-mGFP Cterm S11 Neo Kan. Unique restriction sites shown flanking key modules. The Not I site follows the mGFP S11 stop codon.  enes are cloned with initiator codons just after Nhe I in the multiple cloning site (MCS) and in-frame with the downstream linker and GFP S11 module. The structure of  xpressed proteins of interest (POI) is POI-linker-GFP S11.

 Location of features

• Human cytomegalovirus (CMV) immediate early promoter: 1–589 Enhancer region: 59–465; TATA box: 554–560 Transcription start point: 538 C→G mutation to remove Sac I site: 569
• Multiple cloning site: 613-681 Linker GDGGSGGGS: 682-708
• GFP S11 mouse codon-optimized (GFP amino acids 215-230) GFP S11: 709-756 Stop codon: 757-759
• SV40 early mRNA polyadenylation signal Polyadenylation signals: 918-918 & 942-947 mRNA 3' ends: 951 & 963 Rev. 3.0• f1 single-strand DNA origin: 1010-1465 (Packages noncoding strand of POI-linker-GFP S11.)
• Ampicillin resistance (β-lactamase) promoter –35 region: 1527-1532; –10 region: 1550-1555 Transcription start point: 1562
• SV40 origin of replication: 1806-1941
• SV40 early promoter Enhancer (72-bp tandem repeats): 1639-1710 & 1711-1782 21-bp repeats: 1786-1806, 1807-1827 & 1829-1849 Early promoter element: 1862-1868 Major transcription start points: 1858, 1896, 1902 & 1907
• Kanamycin/neomycin resistance gene Neomycin phosphotransferase coding sequences: Start codon (ATG): 1990-1992; stop codon: 2781-2784
• Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal Polyadenylation signals: 3020-2025 & 3033-3038
• pUC plasmid replication origin: 3369-401

Propagation in E. coli

• Suitable host strains: DH5α, HB101 and other general purpose strains. Single-stranded DNA production requires a host containing an F plasmid such as JM109 or XL1-Blue.
• Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) to E. coli hosts.
• E. coli replication origin: pUC
• Copy number: ~500
• Plasmid incompatibility group: pMB1/Col E1

References

1. Cabantous S, Terwilliger TC, Waldo GS (2005) Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nature Biotech. 23: 102-107.
2. Chun WJ, Waldo GS, Johnson GVW (2007) Split GFP complementation assay: a novel approach to quantitatively measure aggregation of tau in situ: effects of GSK3 beta activation and caspase 3 cleavage. Journal of Neurochemistry 103: 2529-2539.
3. Kozak, M. (1987) Nucleic Acids Res. 15:8125–8148.
4. Gorman, C. (1985) In DNA cloning: A Practical Approach, Vol. II. Ed. D. M. Glover. (IRL Press, Oxford, U.K.), pp. 143–190.