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Parts

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PART NAME
BASIC PARTS
BBa_K2645000 DFP
BBa_K2645001 Green only isoform of DFP
BBa_K2645003 Forward mutant primer
BBa_K2645004 Reverse mutant primer
COMPOSITE PART
BBa_K2645002 DFP cassette

DFP (BBa_K2645000)


Dual Fluorescence Protein is a mutant protein formed due to 8 point mutations of the wild type of Green Fluorescence Protein. It exhibits the characteristic of dual fluorescence by exhibiting red and green fluorescence at 610nm and 540nm respectively.


Green only isoform of DFP (BBa_K2645001)


The Green only isoform of DFP exhibits only green fluorescence due to a single point mutation at the 65th position of the DFP. This point mutation causes DFP to exhibit a single green fluorescence.


Forward mutant primer- DFP to Green only isoform (BBa_K2645003)


This is used to convert the Dual Fluorescent protein into its green only isoform. The primer consists of the position specific (65th position) mutant base and is the primer for the sense strand.


Length (nt.) Tm Duplex Energy at 68 °C Energy Cost of Mismatches
41 78.60
-43.68 kcal/mole 3.51%

Reverse mutant primer- DFP to Green only isoform (BBa_K2645004)


This is used to convert the Dual Fluorescent protein into its green only isoform. The primer consists of the position specific (65th position) mutant base and is the primer for the anti sense strand.


Length (nt.) Tm Duplex Energy at 68 °C Energy Cost of Mismatches
41 78.60
-45.10 kcal/mole 3.49%

DFP cassette (BBa_K2645002)


Dual Fluorescent Protein exhibits fluoresces both green and red at corresponding wavelengths. It is a reporter gene that could be point mutated into green only mutant form using a forward mutant primer and a reverse mutant primer. This protein can be used to screen for the successful occurrence of mutagenesis.

J23101 – Promoter

B0034 – Ribosome Binding Site

K2645000 – DFP

B0010 – Terminator

B0012 – Terminator


DFP CHARACTERISATION


The Absorbance and the Excitation and Emission spectrum of DFP were studied.

ABSORBANCE SPECTRUM

The absorbance peaks for DFP can be seen at two regions within the 300nm-700nm range- the major peak at 395nm and the minor peak at 475nm.

EXCITATION/EMISSION SPECTRUM


FLUORESCENCE COLOR EXCITATION MAXIMUM EMISSION MAXIMUM QUANTUM YIELD OF FLUORESCENCE/span>
Green (DFP) 395nm 508nm 0.75
Red (DFP) 555nm 585nm 0.45

CHARACTERISATION OF DFP CASSETTE




The expression of the reporter gene GFP (shown above) has been characterized with the help of the constitutive promoter (J23101)-RBS (B0034) cassette. Owing to the previously cited studies that have suggested that DFP behaves the same as GFP and holds the same properties, the results for this characterization have been extended to DFP as well.


It can be seen that the growth of the cells remain unaffected by the expression of GFP, as GFP (this interpretation is extrapolated to DFP) is known to be non-toxic to cells.

From the graphs above, it is observed that the expression of the fluorescent protein increases substantially with time.

The information in this page is based on the paper



The First Mutant of the Aequorea Victoria Green Fluorescent Protein That Forms a Red Chromophore†

Alexander S. Mishin,‡,§ Fedor V. Subach,‡,| Ilia V. Yampolsky,§ William King,⊥ Konstantin A. Lukyanov,*,§ and Vladislav V. Verkhusha*,|

Shemiakin-OVchinnikoV Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, Moscow 117997, Russia, and Department of Anatomy and Structural Biology and Flow Cytometry Core Facility, Albert Einstein College of Medicine, Bronx, New York 10461

Received October 23, 2007; Revised Manuscript Received February 27, 2008