evoglow clostridia basic kit product information

Transcription

1 evoglow clostridia basic kit product information

2 glow clostridia basic kit evoglow product information: product cat. no. content plasmids: pglow-c-bs2 FP plasmid pglow-c-pp1 FP plasmid pglow-ck XN -Bs2 FP plasmid pglow-ck XN -Pp1 FP plasmid kits evoglow clostridia basic kit evoglow clostridia expression kit evoglow whole clostridia kit FP FP FP pglow-c-bs2 and pglow-c-pp1 combined in one package pglow-ck XN -Bs2 and pglow-ck XN -Pp1 combined in one package all plasmids for clostridia combined in one kit 3

3 Content General Information 4 Kit Content and Storage Information 4 evoglow Anaerobic Fluorescent Proteins 5 Physical Parameters 5 Instrumental Setup Recommendations 6 Plasmid Maps, Fluorescence Spectra and Sequence Information 7 pglow-c-bs2 7 pglow-c-pp1 9 Contact Information 12 3

4 evocatal GmbH 3

5 Flavin-mononucleotide-based Fluorescent Protein (FbFP) Catalog No. Quantity 20 µg each General Information Fluorescent reporter proteins are valuable noninvasive molecular tools for in vivo real-time imaging of living cells and tissues as well as in vitro fluorescence labeling. A major drawback of common GFP-like reporter proteins is their strict requirement for molecular oxygen as a cofactor for the synthesis of their respective fluorophores. Therefore, they cannot be applied in anaerobic systems (see Fig. 1). The flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) of the evoglow series were developed to overcome these restrictions. The evoglow proteins can be used as fluorescent reporters in both aerobic and anaerobic biological systems and were shown to be functional under completely anoxic conditions, for example in bacterial like R. capsulatus and C. acetobutylicum as well as in the yeast C. albicans (see references). Kit content: The evoglow clostridia basic kit comprises the plasmids pglow-c-bs2 and pglow-c-pp1, which encode for the evoglow reporter proteins evoglow-bs2 and evoglow- Pp1, respectively, whose codon-usage has been adopted exclusively for efficient expression in Clostridia. The genes are cloned SacI/BamHI into the multiple cloning site of puc18 allowing individual subcloning under the control of the promotor of your choice. Origin: The evoglow-c-bs2 and evoglow-c-pp1 genes are based on the evoglow-bs2 and evoglow-pp1 genes, and originate from the Gram-positive bacterium Bacillus subtilis and the Gram-negative bacterium Pseudomonas putida, respectively. For optimal expression of the reporter proteins in clostridia the codonusage has been adopted exclusively. Formulation: pglow plasmids are delivered in aliquots of 20 µg plasmid DNA dissolved in 10 mm Tris/HCl-buffer, ph 8.5. Storage: Plasmids can be stored at - 20 C. Intended use: For research use only. Not for clinical diagnosis. References: Drepper, T. et al. (2007) Nat. Biotechnol. 25: Fig. 1: in vivo-fluorescence of evoglow -proteins compared to that of conventional Yellow Fluorescent Protein (YFP). Pictures by courtesy of Leica Microsystems, Wetzlar. Ernst, F. & Tielker, D. (2008) Cell. Microbiol. 11(2):

6 evoglow - Anaerobic Fluorescent Proteins The designation evoglow comprises a novel type of fluorescent proteins containing a Flavin-mononucleotide-based cofactor. They are thus capable of developing bright cyan-green fluorescence even in the complete absence of oxygen. They fold rapidly in vivo and thus enable immediate detection of expression to study anaerobic interand intracellular processes. Since their fluorescence intensity as well as the excitation and emission spectra of the evoglow proteins lie within the same range as those of other fluorescent proteins, the same instrumental setup can be used for their detection and analysis as for other fluorescent proteins. Recommendations for instruments and filtersets are given in section Instrumental Setup. Physical Parameters: Characteristics evoglow -Bs2 evoglow -Pp1 Fluorescence color cyan-green cyan-green Excitation max. (nm) Emmission max. (nm) Quantum yield ,17 Extinction coefficient (M -1 cm -1 ) ,500 Brightness* pka n.d. n.d. structure dimer dimer Photostability Molecular weight 19 kda ca. 19kDa Amino acids * Brightness quantum yield extinction coefficient

7 Instrumental Setup Recommendations: evoglow proteins can be excellently detected by using the following instrumental setup: microscope: Zeiss Axioplan 1. object lens for fluorescence imaging: Plan APOCHROMAT 100x oil DIC with numerical aperture 1.4 (object lens "Plan-Apochromat" 100x/1.40 Oil DIC) (Immersion oil 518F) filterset 37 (Ex 450/50, BS 480, Em 510/50) lamp: HBO50 camera: Zeiss AxioCam MRm (12 Bit, monochrome) standard imaging software: AxioVision Rel for light microscopy: Zeiss Plan NEOFLUAR 100x oil phase contrast objective (Phase 3) numerical aperture 1.3 (object lens "Plan-Neofluar" 100x/1.30 Oil Ph3) 6

8 pglow-c-bs2 cat. no. Plasmid Map bla (Ap) pglow-c-bs bps NdeI evoglow-bs2-cl glow-c-bs HindIII PstI SalI XbaI BamHI HindIII PstI NdeI SacI EcoRI Restriction Enzyme Analysis Information: Enzyme bp positions: BamHI 429 EcoRI 858 HindIII 399; 444 NdeI 183; 846 PstI 411; 629 SacI 852 SalI 417 XbaI 423 Sequence Landmarks bp positions: rep (pmb1) Ampicillin Resistance evoglow-c-bs2 Gene Origin of Replication EcoRI SacI NdeI BamHI XbaI SalI >> tac gaa ttc gag ctc cat atg......taa gga tcc tct aga gtc gac ctg cag gca tg...>> 5 evoglow-c-bs2 3 >> atg ctt aag ctc gag gta tac......att cct agg aga tct cag ctg gac gtc cgt ac...>> HincII Limitation of use: The plasmids distributed by company evocatal GmbH under the trade mark evoglow are subject to intellectual property right applications. Company evocatal GmbH commits the plasmids and genes to the costumer solely for the purpose of own scientific, non-commercial investigation or own, non-commercial research and development purposes. Any commercial use requires the conclusion of a separate license contract. For commercial customers (i.e. companies or any for-profit-organisations) the permission of use for the above said research purposes of the evoglow genes and plasmids is limited to six (6) months, starting from the date of purchase. For detailed license information please contact evocatal GmbH, Germany. Furthermore, the modification of the evoglow-gene-sequences and of the plasmids is assertive prohibited. By the purchase of the plasmids without notice of defect within a term of 14 days beginning with the day of delivery, the customer agrees to the limitation of use above. Exclusion of liability: Company evocatal GmbH commits the plasmids distributed under the trade mark evoglow to the customer solely for use in microorganism. The plasmids are not intended for the use in human medical or veterinarian medical diagnostic purposes, or in the animal or human body; any liability of the company evocatal GmbH is excluded. The committed plasmids are classified to security level S1. The national safety regulations for handling genetic engineered microorganism have to be considered and fulfilled when handling the plasmids. By the purchase of the plasmids without notice of defect within a term of 14 days beginning with the day of delivery, the customer agrees to the above exclusion of liability and indemnifies company evocatal GmbH from any liability for the use of the plasmids beyond the intended application. Sequence Information: 7

9 Fluorescence Spectra: Technical Data: excitation max.: emission max.: 449 nm 495 nm (λ ex : 450 nm) molar extinction coefficient (FMN): ε = 12,500 ±500 M -1 cm -1 (at 450 nm) quantum yield: Q F = 0,39 Sequence Information: evoglow-c-bs2 nucleotide sequence (414 bp) evoglow-bs2 amino acid sequence (137 aa) 1 ATGGCAAGTT TTCAAAGTTT TGGAATACCT 31 GGACAACTTG AAGTTATAAA AAAAGCACTT 61 GATCATGTTA GAGTTGGAGT TGTTATAACT 91 GATCCTGCAT TAGAAGATAA TCCAATAGTT 121 TATGTTAATC AAGGATTTGT TCAGATGACA 151 GGATATGAAA CAGAAGAAAT ATTAGGAAAA 181 AATGCAAGAT TTCTTCAAGG TAAACATACA 211 GATCCTGCAG AAGTTGATAA TATAAGAACA 241 GCATTACAGA ATAAAGAACC TGTTACAGTT 271 CAAATACAAA ATTATAAGAA AGATGGAACT 301 ATGTTTTGGA ATGAACTTAA TATAGATCCT 331 ATGGAAATAG AAGATAAGAC ATATTTTGTT 361 GGAATACAGA ATGATATAAC AAAGCAAAAA 391 GAATATGAAA AGCTTCTTGA ATAA 1 MASFQSFGIP GQLEVIKKAL DHVRVGVVIT 31 DPALEDNPIV YVNQGFVQMT GYETEEILGK 61 NARFLQGKHT DPAEVDNIRT ALQNKEPVTV 91 QIQNYKKDGT MFWNELNIDP MEIEDKTYFV 121 GIQNDITKQK EYEKLLE 8

10 pglow-c-pp1 cat. no. Plasmid Map Restriction Enzyme Analysis Information: bla (Ap) NdeI evoglow-pp1-cl glow-c-pp pglow-c-pp bps HindIII PstI SalI XbaI BamHI NdeI SacI EcoRI Enzyme bp positions: BamHI 429 EcoRI 858 HindIII 399; 444 NdeI 183; 879 PstI 411 SacI 885 SalI 417 XbaI 423 Sequence Landmarks bp positions: rep (pmb1) Ampicillin Resistance evoglow-c-bs2 Gene Origin of Replication EcoRI SacI NdeI BamHI XbaI SalI >> tac gaa ttc gag ctc cat atg......taa gga tcc tct aga gtc gac ctg cag gca tg...>> 5 evoglow-c-pp1 3 >> atg ctt aag ctc gag gta tac......att cct agg aga tct cag ctg gac gtc cgt ac...>> HincII PstI Limitation of use: The plasmids distributed by company evocatal GmbH under the trade mark evoglow are subject to intellectual property right applications. Company evocatal GmbH commits the plasmids and genes to the costumer solely for the purpose of own scientific, non-commercial investigation or own, non-commercial research and development purposes. Any commercial use requires the conclusion of a separate license contract. For commercial customers (i.e. companies or any for-profit-organisations) the permission of use for the above said research purposes of the evoglow genes and plasmids is limited to six (6) months, starting from the date of purchase. For detailed license information please contact evocatal GmbH, Germany. Furthermore, the modification of the evoglowgene-sequences and of the plasmids is assertive prohibited. By the purchase of the plasmids without notice of defect within a term of 14 days beginning with the day of delivery, the customer agrees to the limitation of use above. Exclusion of liability: Company evocatal GmbH commits the plasmids distributed under the trade mark evoglow to the customer solely for use in microorganism. The plasmids are not intended for the use in human medical or veterinarian medical diagnostic purposes, or in the animal or human body; any liability of the company evocatal GmbH is excluded. The committed plasmids are classified to security level S1. The national safety regulations for handling genetic engineered microorganism have to be considered and fulfilled when handling the plasmids. By the purchase of the plasmids without notice of defect within a term of 14 days beginning with the day of delivery, the customer agrees to the above exclusion of liability and indemnifies company evocatal GmbH from any liability for the use of the plasmids beyond the intended application. 9

11 Fluorescence Spectra: Technical Data: excitation max.: emission max.: 449 nm 495 nm (λ ex : 450 nm) molar extinction coefficient (FMN): ε = 12,500 ±500 M -1 cm -1 (at 450 nm) quantum yield: Q F = 0,17 Sequence Information: evoglow-c-pp1 nucleotide sequence (447 bp) evoglow-pp1 amino acid sequence (148 aa) 1 ATGATAAATG CAAAACTTCT TCAGCTTATG 31 GTTGAACATA GTAATGATGG AATAGTTGTT 61 GCAGAACAGG AAGGTAACGA AAGTATACTT 91 ATATATGTAA ATCCTGCATT TGAAAGATTA 121 ACAGGATATT GCGCAGATGA TATACTTTAT 151 CAAGATGCTA GATTTTTACA GGGTGAAGAT 181 CATGATCAAC CTGGAATAGC AATAATAAGA 211 GAAGCAATAA GAGAAGGTAG ACCTTGTTGT 241 CAGGTTTTAA GAAATTATAG AAAGGATGGA 271 AGTTTATTTT GGAATGAACT TAGTATAACA 301 CCTGTTCATA ATGAAGCAGA TCAACTTACT 331 TACTATATAG GAATACAAAG AGATGTTACA 361 GCACAAGTTT TTGCAGAAGA AAGAGTTAGA 391 GAATTAGAAG CAGAAGTTGC AGAATTAAGA 421 AGACAACAAG GACAAGCAAA ACATTAA 1 MINAKLLQLM VEHSNDGIVV AEQEGNESIL 31 IYVNPAFERL TGYCADDILY QDARFLQGED 61 HDQPGIAIIR EAIREGRPCC QVLRNYRKDG 91 SLFWNELSIT PVHNEADQLT YYIGIQRDVT 121 AQVFAEERVR ELEAEVAELR RQQGQAKH 11

12 Notes: 10

13 Contact Information: For any further information, questions or remarks please contact: Jena Bioscience GmbH Loebstedter Strasse 80 D Jena Germany phone: fax: