evoglow

basic kit
product information

Cat. No.: 2.1.010

Flavin-mononucleotide-based Fluorescent Protein (FbFP)

evoglow
Catalog No. evo-2.1.010

basic kit
Quantity 20 g 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 existing GFP-like reporter proteins
is their strict requirement for molecular oxygen as a
cofactor for the synthesis of their respective
chromophores. Therefore, the application in anaerobic
systems is not possible (see Fig. 1).
The flavin mononucleotide (FMN)-based fluorescent
proteins (FbFPs) from 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.

Kit content: The evoglow basic kit contains the

plasmids pGLOW-Bs1, pGLOW-Bs1-stop, pGLOW-Bs2,
pGLOW-Bs2-stop, pGLOW-Pp1 and pGLOW-Pp1-stop.
The genes are cloned SacI/PstI or SacI/BamHI into the
multiple cloning site of pUC18 for individual subcloning.
Each gene is available in a fusion-version - without stop
codon - and in an expression-version containing the
TGA stop codon.

Origin: The evoglow-Bs1 and Bs2 genes were cloned

from the Gram positive bacterium Bacillus subtilis. The
Bs1-sequence is the wild-type codon-usage, whereas
Bs2-sequence has been adjusted to Escherichia coli
codon usage and is truncated, i.e. comprises the LOVdomain only. The evoglow-Pp1 gene was cloned from
the Gram negative bacterium Pseudomonas putida.

Formulation: pGLOW plasmids are delivered as 20 g

plasmid DNA dissolved in regular 10 mM Tris/HClbuffer, pH 8,5.

Storage: pGLOW plasmids can be stored at -20C.

Intended use: For research use only. Not for clinical

diagnosis.

Fig. 1: in vivo-fluorescence of evoglow-proteins compared

to that of conventional Yellow Fluorescent Protein (YFP).

References: Drepper, T. et al. (2007) Nat. Biotechnol.

25: 443-445.

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 inter- and 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-Bs1

evoglow-Bs2

evoglow-Pp1

cyan-green

cyan-green

cyan-green

Excitation max. (nm)

450

450

450

Emmission max. (nm)

495

495

495

0,4

0,39

0,17

12,500

12,500

12,500

4.9

2.1

n.d.

n.d.

n.d.

monomer

dimer

dimer

++

+++

ca. 33kDa

ca. 19kDa

ca. 19kDa

262

137

148

Fluorescence color

Quantum yield
-1

-1

Extinction coefficient (M cm )
Brightness*
pKa
structure
Photostability
Molecular weight
Amino acids

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 l DIC mit numerical aperature 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. 4.6.

for light microscopy: Zeiss Plan NEOFLUAR 100x l phase contrast objective (Phase 3) numerical aperature 1,3
(object lens "Plan-Neofluar" 100x/1,30 Oil Ph3)

evoglow basic kit

pGLOW-Bs1 and pGLOW-Bs1-stop

Plasmid Information

Plasmid Map
Alw44I
NdeI

restriction analysis information:

HincII
Sal I
Xb aI
BamHI

Alw 44I

enzyme:

fragment size (bp)

Alw44I

further restriction sites at positions (bp)

evoglow-Bs1
evoglow-Bs
1

bla (Ap)

497
1246
1720

EcoRI
HindIII
NdeI
PstI

pGLOW-Bs1
pGLOW-Bs1
3463 bp

3463 bps

851
399
183
411

1227
813
1215
998

sequence landmarks (bp positions):

lac

ampicillin-resistence:
rep (pMB1):
lac-promotor:
evoglow-Bs1-gene:

NdeI
Sac I

rep(pMB1)

3263-2403
2243-1629
1323- 284
1217- 435

Alw 44I

HincII
EcoRI

SacI

NdeI

>> tac gaa ttc gag ctc cat atg...

>> atg ctt aag ctc gag gta tac...

BamHI
evoglow-Bs1

XbaI

SalI

PstI

...atg gga tcc tct aga gtc gac ctg cag gca tg...>>
...tac cct agg aga tct cag ctg gac gtc cgt ac...>>

In pGLOW-Bs1-stop the evoglow-Bs1-gene includes a termination codon indicated in red

HincII
EcoRI

SacI

NdeI

>> tac gaa ttc gag ctc cat atg...

evoglow-Bs1-stop
>> atg ctt aag ctc gag gta tac...

BamHI

XbaI

SalI

PstI

...atg tga gga tcc tct aga gtc gac ctg cag gca tg...>>
...tac act cct agg aga tct cag ctg gac gtc cgt ac...>>

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 to the costumer solely for the purpose of own scientific investigation or own research and development purposes. Any commercial use requires the conclusion of a separate
license contract. Furthermore, the modification of the Bs1-FbFP sequence (evoglow-Bs1) 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.

evoglow basic kit

Fluorescence Spectra:
400
700

Fluorescence Intensity (a.u.)

650

450

Technical Data:
500

550
700

evoglow-Bs1 Excitation
excitation
YtvA-C62A
evoglow-Bs1 Emission
emission
YtvA-C62A

600

600

550

550

500

500

450

450

400

400

350

350

300

300

250

250

200

200

150
400

450

excitation max.:

449 nm

emission max.:

495 nm (ex: 450 nm)

650

500

molar extinction
coefficient (FMN):

quantum yield:

-1

= 12,500 500 M cm
(at 450 nm)

-1

QF = 0,38

150
550

wavelength (nm)

Sequence Information:
evoglow-Bs1 nucleotide sequence (786 bp)
1
31
61
91
121
151
181
211
241
271
301
331
361
391
421
451
481
511
541
571
601
631
661
691
721
751
781

ATGGCTAGTT
GGACAGCTGG
GATCACGTGC
GATCCCGCAC
TACGTAAATC
GGCTACGAGA
AACGCACGCT

TTCAATCATT TGGGATACCA
AAGTCATCAA AAAAGCACTT
GAGTCGGTGT GGTAATTACA
TTGAAGATAA TCCTATTGTC
AAGGCTTTGT TCAAATGACC
CCGAGGAAAT TTTAGGAAAG
TCTTACAGGG GAAACACACA
GATCCTGCAG AAGTGGACAA CATCAGAACC
GCTTTACAAA ATAAAGAACC GGTCACCGTT
CAGATCCAAA ACTACAAAAA AGACGGAACG
ATGTTCTGGA ATGAATTAAA TATTGATCCA
ATGGAAATAG AGGATAAAAC GTATTTTGTC
GGAATTCAGA ATGATATCAC CAAGCAAAAA
GAATATGAAA AGCTTCTCGA GGATTCCCTC
ACGGAAATTA CTGCACTTTC AACTCCTATT
GTCCCGATTC GCAATGGCAT TTCGGCTCTT
CCGCTAGTCG GAAACCTGAC AGAGGAGCGA
TTTAATTCCA TCGTTTGCAC ATTGACGAAT
ATCTTATCAA CATCCAAAGA TGATTATTTG
ATCATTGATT TATCCGGATT GGCCCAAGTG
AACGAACAAA CGGCCGACCA AATTTTCAAG
CTGAGCCATT TGCTGAAATT GACCGGAACT
GAGTTAATCA TTACTGGCAT TAAGCCTGAA
TTGGCTATGA AAATGAATAA ACTGGATGCC
AATTTTTCGT CGCTGAAAAC ATATTCAAAT
GTAAAGGATG CCGTTAAAGT GCTTCCGATT
ATG TGA term. codon in pGLOW-Bs1-stop only

evoglow-Bs1 amino acid sequence (261 aa)

1
31
61
91
121
151
181
211
241

MASFQSFGIP
DPALEDNPIV
NARFLQGKHT
QIQNYKKDGT
GIQNDITKQK
VPIRNGISAL
ILSTSKDDYL
LSHLLKLTGT
NFSSLKTYSN

GQLEVIKKAL
YVNQGFVQMT
DPAEVDNIRT
MFWNELNIDP
EYEKLLEDSL
PLVGNLTEER
IIDLSGLAQV
ELIITGIKPE
VKDAVKVLPI

DHVRVGVVIT
GYETEEILGK
ALQNKEPVTV
MEIEDKTYFV
TEITALSTPI
FNSIVCTLTN
NEQTADQIFK
LAMKMNKLDA
M

Note:
The nucleotide sequence of evoglow-Bs1-stop is identical
to that of evoglow-Bs1 but includes a termination-codon
at the 3-end as indicated in the adjacent sequence.
The plasmid base pair numbering is hence shifted 3
positions in the stop-constructs.
All plasmids in this kit are derived from pUC18 and
replicate only in E. coli and closely related microorganisms.
Plasmids are not suitable for expression.

evoglow basic kit

pGLOW-Bs2 and pGLOW-Bs2-stop

Plasmid Information

Plasmid Map

Alw 44I
NdeI
restriction analysis information:

HindIII
Pst I
Xho I

Alw 44I

enzyme

fragment size (bp)

Alw44I

evoglow-Bs2

bla (Ap)

further restriction sites at (bp)

evoglow-Bs2

EcoRI
HindIII
NdeI
PstI
SacI
XhoI

pGLOW-Bs2
3076 bp

3076 bps

lac

497
1246
1331

NdeI
Sac I
Eco RI

851
399
828
411
834
420

183

sequence landmarks (bp):

ampicillin-esistence:
rep (pMB1):
lac-promotor:
evoglow-Bs2-gene:

rep(pMB1)

2876-2016
1856-1242
936- 897
830- 417

Alw 44I

EcoRI

SacI

NdeI

>> tac gaa ttc gag ctc cat atg...

5 >> atg ctt aag ctc gag gta tac...

PstI
evoglow-Bs2

SphI

HindIII

...gag gct gca ggc atg caa gct tgg...>>

...ctc gga cgt ccg tac gtt cga acc...>>

In pGLOW-Bs2-stop the evoglow-Bs2-gene includes a termination codon indicated in red

EcoRI

SacI

NdeI

>> tac gaa ttc gag ctc cat atg...

>> atg ctt aag ctc gag gta tac...

PstI
evoglow-Bs2

SphI

HindIII

...gag tga ctg cag gca tgc aag ctt ggc...>>

...ctc act gac gtc cgt acg ttc gaa ccg...>>

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 to the costumer solely for the purpose of own scientific investigation or own research and development purposes. Any commercial use requires the conclusion of a separate
license contract. Furthermore, the modification of the Bs2-FbFP sequence (evoglow-Bs2) 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.

evoglow basic kit

Fluorescence Spectra:

Technical Data:

excitation max.:

449 nm

emission max.:

495 nm (ex: 450 nm)

molar extinction
coefficient (FMN):

quantum yield:

-1

= 12,500 500 M cm
(at 450 nm)

-1

QF = 0,39

Sequence Information:

evoglow-Bs2 nucleotide sequence (411 bp)

1
31
61
91
121
151
181
211
241
271
301
331
361
391

ATGGCGTCGT
GGCCAGCTGG
GATCACGTGC
GATCCCGCGC
TACGTGAACC
GGCTACGAGA
AACGCGCGCT

TCCAGTCGTT CGGCATCCCG
AAGTCATCAA GAAGGCGCTG
GCGTCGGCGT GGTCATCACC
TGGAAGATAA CCCGATCGTC
AGGGCTTCGT GCAGATGACC
CCGAGGAAAT CCTGGGCAAG
TCCTCCAGGG GAAGCACACC
GATCCGGCGG AAGTGGACAA CATCCGCACC
GCGCTGCAAA ATAAAGAACC GGTCACCGTG
CAGATCCAGA ACTACAAGAA GGACGGCACG
ATGTTCTGGA ACGAACTGAA CATCGATCCG
ATGGAAATCG AGGATAAGAC GTATTTCGTC
GGCATCCAGA ACGACATCAC CAAGCAGAAG
GAATATGAAA AGCTGCTCGA G TGA
termination codon in pGLOW-Bs2-stop only

evoglow-Bs2 amino acid sequence (137 aa)

1
31
61
91
121

MASFQSFGIP
DPALEDNPIV
NARFLQGKHT
QIQNYKKDGT
GIQNDITKQK

GQLEVIKKAL
YVNQGFVQMT
DPAEVDNIRT
MFWNELNIDP
EYEKLLE

DHVRVGVVIT
GYETEEILGK
ALQNKEPVTV
MEIEDKTYFV

Note:
The nucleotide sequence of evoglow-Bs2-stop is identical
to that of evoglow-Bs2 but includes a termination-codon
at the 3-end as indicated in red in the adjacent sequence.
The plasmid base pair numbering is hence shifted 3
positions in the stop-constructs.
All plasmids in this kit are derived from pUC18 and
replicate only in E. coli and closely related microorganisms.
Plasmids are not suitable for expression.

evoglow basic kit

pGLOW-Pp1 and pGLOW-Pp1-stop

Plasmid Information

Plasmid Map
Alw 44I
NdeI

restriction analysis information:

Alw 44I

HindIII
Pst I

enzyme:

fragment size (bp)

Alw44I

376
497
988
1246

Alw 44I

evoglow-Pp
evoglow-Pp1
1

bla (Ap)

further restriction sites at (bp)

EcoRI
HindIII
NdeI
PstI
SacI

pGLOW-Pp1
3107 bp

lac

NdeI
Sac I
Eco RI

871
399
183 859
411
865

sequence landmarks (bp positions):

rep(pM B1)

ampicillin-resistence:
rep (pMB1):
lac-promotor:
evoglow-Pp1-gene:

Alw 44I

EcoRI

SacI

NdeI

>> tac gaa ttc gag ctc cat atg...

>> atg ctt aag ctc gag gta tac...

PstI
evoglow-Pp1

SphI

2907-2047
1887-1273
967- 928
861- 418

HindIII

...cac gct gca ggc atg caa gct tgg c...>>

...gtg cga cgt ccg tac gtt cga acc g...>>

In pGLOW-Bs2-stop the evoglow-Bs2-gene includes a termination codon indicated in red

EcoRI

SacI

NdeI

>> tac gaa ttc gag ctc cat atg...

>> atg ctt aag ctc gag gta tac...

PstI
evoglow-Pp1

SphI

HindIII

...cac tga ctg cag gca tgc aag ctt ggc...>>

...gtg act gac gtc cgt acg ttc gaa ccg...>>

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 to the costumer solely for the purpose of own scientific investigation or own research and development purposes. Any commercial use requires the conclusion of a separate
license contract. Furthermore, the modification of the Pp1-FbFP sequence (evoglow-Pp1) 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.

evoglow basic kit

Fluorescence Spectra:

Technical Data:

excitation max.:

449 nm

emission max.:

495 nm (ex: 450 nm)

molar extinction
coefficient (FMN):

quantum yield:

-1

= 12,500 500 M cm
(at 450 nm)

-1

QF = 0,17

Sequence Information:

evoglow-Pp1 nucleotide sequence (444 bp)

evoglow-Pp1 amino acid sequence (148 aa)

1 ATGATCAACG

CAAAACTCCT

GCAACTGATG

1 MINAKLLQLM

VEHSNDGIVV

AEQEGNESIL

31 GTCGAACATT

CCAACGATGG

CATCGTTGTC

31 IYVNPAFERL

TGYCADDILY

QDARFLQGED

61 GCCGAGCAGG

AAGGCAATGA

GAGCATCCTT

61 HDQPGIAIIR

EAIREGRPCC

QVLRNYRKDG

91 ATCTACGTCA

ACCCGGCCTT

CGAGCGCCTG

91 SLFWNELSIT

PVHNEADQLT

YYIGIQRDVT

121 ACCGGCTACT

GCGCCGACGA

121 AQVFAEERVR

ELEAEVAELR

RQQGQAKH

151 CAGGACGCCC

GTTTTCTTCA

TATTCTCTAT
GGGCGAGGAT

Note:

181 CACGACCAGC

CGGGCATCGC

AATTATCCGC

211 GAGGCGATCC

GCGAAGGCCG

CCCCTGCTGC

241 CAGGTGCTGC

GCAACTACCG

CAAAGACGGC

271 AGCCTGTTCT

GGAACGAGTT

GTCCATCACA

301 CCGGTGCACA

ACGAGGCGGA

CCAGCTGACC

331 TACTACATCG

GCATCCAGCG

CGATGTCACA

361 GCGCAAGTAT

TCGCCGAGGA

AAGGGTTCGC

All plasmids in this kit are derived from pUC18 and

replicate only in E. coli and closely related microorganisms.

391 GAGCTGGAGG

CTGAAGTGGC

GGAACTGCGC

Plasmids are not suitable for expression.

421 CGGCAGCAGG

GCCAGGCCAA

GCAC TGA

The nucleotide sequence of evoglow-Pp1-stop is identical

to that of evoglow-Pp1 but includes a termination-codon
at the 3-end as indicated in red in the adjacent sequence.
The plasmid base pair numbering is hence shifted 3
positions in the stop-constructs.

termination codon in pGLOW-Pp1-stop only

Contact Information:
For any further information, questions or remarks please contact:

evocatal GmbH
Merowingerplatz 1a
40225 Dsseldorf
www.evocatal.com
[email protected]

BioCat GmbH
Im Neuenheimer Feld 584
D-69120 Heidelberg
www.biocat.com
[email protected]

10