Team:REC-CHENNAI/Parts
| 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.
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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.
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