The best green emitting luciferase variant for dual color reporter assays is GR7', which has a thermostability of 12.8 hours, a green to red ratio of 24:1, and low red background of 3%. Future experiments will analyze the thermostability of a new point mutation, F465R, introduced into GR7'.

1. Conclusions and Future Directions
Currently the best green emitting enzyme for the dual color reporter assay is GR7’,which is
derived from GR4’ with three additional mutations: I232A/F295L/E354K. This enzyme has a
thermostability of 12.8 hours, a green to red ratio of 24:1 and a low red background of only 3%.
The thermostability of a new point mutation F465R, that was introduced into GR7’ will be
analyzed in future experiments.
Luciferase Variants for Dual Color Reporter Assays & Bioluminescence Imaging
Munya Talukder, Danielle Fontaine,
Curran E. Behney, Tara L. Southworth and Bruce R. Branchini
Department of Chemistry, Connecticut College, New London, Connecticut 06320
Background
Luciferase from the firefly Photinus pyralis (Ppy) is a naturally occurring enzyme that emits yellow-green light
(557nm) as a result of an oxidation reaction with oxygen, Mg-ATP and the substrate luciferin.
Dual Color Reporter Assay
Dual color reporter assay is an important tool in the study of eukaryotic gene expression and regulation. It is
utilized to assess the ability of promoters to enhance gene expression. For this technique it is desirable to have
the following:
1. Two human codon-optimized luciferase variants that express well in mammalian cells.
.
2. A single solution containing two bioluminescence producing substrates LH2 and BtLH2.
Acknowledgements
Many thanks to Professor Bruce Branchini for this amazing opportunity and Danielle Fontaine for
her guidance. Also the Bioluminescence Research Group of 2014: Curran E. Behney, Tara L.
Southworth, Leah Salituro and Aliya Holland.
This work was supported by the Sherman Fairchild and NSF foundations.
Abstract
North American firefly Photinus pyralis Luciferase is an enzyme widely utilized for a variety of application such as
gene reporter assays and in vivo imaging. Red and green emitting luciferase variants can be utilized simultaneously
for dual-color reporter assays or can be used individually for in vivo bioluminescence imaging (BLI). In order to
enhance dual color reporter gene applications, mutagenesis experiments were conducted to engineer two human
codon-optimized (HCO) luciferase variants that are similar in terms of activity, thermostability and selectivity to their
non-optimized counterparts. Residue changes I232A/F295L/E354K were sequentially introduced into the best
green emitting luciferase variant GR4’(T214A/F250T/Y340F/L291I) to improve thermostability at 37oC while
retaining satisfactory specific activity and selectivity towards BtLH2. The residue E354K was reverted to K354E to
decrease the thermostability of the red enzyme in order to correspond to that of GR4’. The results of this present
research suggest that these novel luciferase variants will significantly enhance the performance of dual-color
reporter assays in mammalian cells by effectively separating the two bioluminescent signals while maintaining
sensitivity.
References
1. Branchini BR, Ablamsky DM, Davis AL, Southworth TL, Butler B, Fan F, Jathoul AP, Pule MA.
Red-emitting luciferases for bioluminescence reporter and imaging applications. Anal
Biochem 2010 1/15;396(2):290-7.
2. Branchini BR, Ablamsky DM, Murtiashaw MH, Uzasci L, Fraga H, Southworth TLThermostable
red and green light-producing firefly luciferase mutants for bioluminescent reporter
applications. Anal Biochem 2007 2/15;361(2):25362.
Red-emitting LuciferaseGreen-emitting Luciferase
Project Objectives
I. Engineer two luciferase variants so they are similar in terms of activity and protein thermostability at 37oC
 Red-emitting luciferase should react with LH2
 Green-emitting luciferase should have a greater affinity for BtLH2 which would blue shift its emission signal causing
the spectrum overlap to decrease.
II. Improve the poor signal separation between the two bioluminescent signals generated by the green- emitting
luciferase and red- emitting luciferase.
Figure 1. Normalized bioluminescence emission spectra at pH 7.8. The red and green shadings indicate the spectral regions transmitted through the green
(Kodak Wratten 44A) and red (Kodak Wratten 29) filters.
Results
Table 1: Properties of Wild-type luc and luc variants at pH 7.8 with LH2 and BtLH2
Enzyme
LH2
Relative Specific Activity a
BtLH2
Relative Specific Activity a
Bioluminescence Emission
max
b
Flash
Height
2min
Integral
30s delay, 30s
integral
% decay
at 2min
Flash
Height
2min
Integral
30s delay,
30s integral
% decay
at 2min LH2 BtLH2 LH2/BtLH2
Ppy WT 164 000 3.1 x 10 7
4.9 x 106
97 3 660 8.4 X 106
2.3 x 106
16 560 (70) 524 (57) -
GR4’/I232A/F295L/E354K
(GR7′)
31 532 2.0 x 107
4.6 x 106
80 8 621 2.2 x 107
5.8 x 106
21 555 (88,134) 520 535 (91,143)
GR7′/F465R 38 723 2.1 x 107
4.6 x 106
86 7 372 1.8 x 107
4.7 x 106
22 551(75,120) 518 (62) 538 (75,131)
GR7′/E488G 52 206 2.9 x 107
6.5 x 106
85 10 217 2.3 x 107
5.8 x 106
23 553 (79,122) 519 (62) 539 (82,132)
GR7′/A532T 35 329 2.8 x 107
6.5 x 106
80 9 118 2.0 x 107
5.2 x 106
27 548 (78,125) 518 (61) 536 (81,131)
a
Specific activities were obtained at pH 7.8 with either LH2 or BtLH2 (350 M) and Mg-ATP (2 mM). Integrated activities are based on total light emission in 2 min. b
Bioluminescence emission maximum at pH 7.8 with the bandwidth at both full-width half-maximum (50%) and 20% shown in parentheses. Emission maxima were obtained from two
trials and the standard deviation is ± 1 nm.
Enzyme-Substrate kcat Km (μM)
kcat/Km (mM-
1s-1)
Ratio
LH2:BtLH2
(kcat/Km)
PpyWT-LH2 0.29 17 17 15.50
PpyWT-BtLH2 0.006 5.6 1.1
GR4’/I232A/F295L/E354K (GR7′)-LH2 0.04 85 0.47 0.56
GR4’/I232A/F295L/E354K (GR7′)-BtLH2 .01 13 .81
GR7′/F465R-LH2 0.0495 38 1.3
GR7′/F465R-BtLH2 0.0094 9 1.05 1.24
GR7′/E488G-LH2 0.0665 46 1.44
GR7′/E488G-BtLH2 0.0142 12 1.18 1.22
GR7′/A532T-LH2 0.044 51 0.86
GR7′/A532T-BtLH2 0.012 10 1.23 0.69