SuperFolder GFP Expression Plasmid Product

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

SuperFolder GFP is a highly engineered robustly folded version of GFP that shows greater tolerance to chemical denaturants and extreme temperatures with improved folding kinetics. The SuperFolder GFP DNA allows a test protein to be expressed as an N-terminal fusion with SuperFolder GFP. SuperFolder GFP fluorescence is naffected by the fusion 1 partner misfolding or solubility and is directly proportional to the amount of expressed protein.

REAGENTS

Components Supplied:
SuperFolder GFP Plasmid in TE buffer: 100ng store at -20^oC.
Note: The stability of the components included in this kit is approximately 6 months when stored at -20^oC. 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:

A. Preparation of Insert DNA:

• Prior to performing the assay, carefully read all instructions.
1. Perform plasmid prep and/or PCR. Use standard materials/protocol not included.
2. Using standard protocol, create a 3’Nde1 sticky end.
3. Optional: Purify digest fragment from agarose gel.

B. Preparation of SuperFolder DNA:

1. To ensure that you have a renewable source of plasmid DNA, transform the plasmid vector in an 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 cloning using Plasmid Preparation kits or other techniques (not included).
3. Perform restriction enzyme digest of the SuperFolder plasmid using Nde1 and BamH1 to excise and prepare the SuperFolder DNA. Follow the manufacturer’s instructions for use of the enzymes. Leave sticky ends in preparation for ligation
4. Optional: Dephosphorylate the digest to decrease non-recombinant background. Use molecular grade calf intestinal or shrimp alkaline phosphatase according to the manufacturer’s directions.
5. Perform ligation reaction according to manufacturers’ instructions.
6. Store DNA at –20^o C until used.

 C. Clone DNA Insert as an N-terminal Fusion into a Secondary Vector:

1. Ligate the DNA insert with the digested SuperFolder DNA using standard DNA ligation protocol and manufacturer’s protocol resulting in a plasmid containing the SuperFolder fusion. 
2. Transform the SuperFolder-fusion plasmid in an expression host for high yields of quality plasmid. Use standard methods based on the screening host used. 
3. Identify the SuperFolder- positive clones using standard methods. Note: IPTG / X-gal screening is effective in the first 24 hours post plating as the T7 promoter is highly active and absorbs resources from the LacZ gene (positive colonies will fluoresce under long wavelength UV light). 
4. Perform plasmid DNA purification, sequence to verify reading frame, or use in vitro transcription/translation.

D. Detection:

1. SuperFolder fluoresces at 490nm excitation with emission at 510nm.
2. Live cultures can be directly observed, by direct fluorescence of colonies, by microscopy or by flow cytometry.
3. Purified protein fusions can be detected by fluorimiter, a fluorescent plate reader or by fluorescence spectrometer.

SEQUENCE INFORMATION

• Detailed sequence information is available on request.

SuperFolder GFP Plasmid

• Flanking Sequences of SuperFolder GFP C6HIS and internal restriction sites.
• Engineering and characterization of SuperFolder GFP is described in: Pedelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS (2006) Engineering and characterization of a SuperFolder green fluorescent protein. Nature Biotechnology 24: 79-88.

Flanking sequences:

REFERENCES
Pédelacq et. al. “Engineering and characterization of a SuperFolder green fluorescent protein,” Nature Biotechnology 24, 79 - 88 (2005)
Cava et. al. “Expression and use of SuperFolder green fluorescent protein at high temperatures in vivo: a tool to study extreme thermophile biology.” J Environmental microbiology 10(3):605 2008 Mar.
Andrews et. al. “The Rough Energy Landscape of SuperFolder GFP Is Linked to the Chromophore,” Journal of Molecular Biology, Volume 373, Issue 2, 19 October 2007, Pages 476-490
Waldo et al. "Rapid protein-folding assay using green fluorescent protein," Nature Biotechnology 17, 691 - 695, July 1999.
Cabantous et. al. “New molecular reporters for rapid protein folding assays.” PLoS ONE 3(6) 2008