Difference between revisions of "Part:BBa K1732005"

Revision as of 00:02, 17 September 2015

J23100-B0034-Renilla-B0015

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 832
Illegal XhoI site found at 641
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 265
1000
COMPATIBLE WITH RFC[1000]

This sequence has been codon optimized for E. coli and is used to produce optimal amounts of Renilla luciferase so that it can be used to compare the luminescence as measured by standard luminometers as well as other DIY luminometers.

pSB1C3 backbone (part:pSB1C3) with J23100 promoter (BBa_J23100) and RBS (BBa_B0034), Renilla (BBa_52008), and B0015 terminator (BBa_B0015).

Renilla Luciferase, also known as Rluc, is a protein that emits light when coelenterazine is added. Rluc can be used as a reporter to track other proteins or to monitor the activity of a promoter (iGEM 2006_Slovenia).

Bioluminescent Mechanism of Coelenterazine

[2]

Coelenterazine is found in several aquatic organisms and is a luminescent substrate for many luciferase enzymes. For this study, Renilla Luciferase interacted with coelenterazine to determine relative light output and kinetics [3].

In the mechanism, coelenterazine reacts with oxygen to yield 1,2-dioxetane (compound 2). Subsequent loss of carbon dioxide leads to the intermediate, followed by emission of a photon.

The image above is the chemiluminescent mechanism of coelenterazine. The bioluminescent mechanism is similar, except that the excited state molecule in bioluminescent is phenolate anion instead of the amide anion [2].

Before running experiments with Renilla Luciferase, the location of the luciferase in the media was determined by comparing the light output of overnight grown culture of Renilla cells with those of the pellet and the supernatant. The enilla culture was spun down, and the pellet represented intracellular localization while the supernatant represented extracellular localization.

It was expected that Renilla Luciferase was localized in the cell, and the level of light output from the pellet matched that of the overnight culture and was significantly higher than that of the media. This suggested that the luciferase is located intracellularly.

The two types of competent cells used were Mach and Top10 cells. Mach1 cells are one of the fastest growing competent strain and Top10 cells also have transformation and cloning efficiency. Both are able to replicate high number of plasmids at stable levels.

From the graphs, it can be determined that higher concentrations of coelenterazine allows for a higher light output. However, the decay rate is not as significant as that of PelB Gaussia at higher concentration of coelenterazine. In addition, the plateau level steadies at a higher light output when given higher concentrations of coelenterazine. It is also important to note that the plateau occurs quickly.

@@ Line 31: / Line 31: @@
 Coelenterazine is found in several aquatic organisms and is a luminescent substrate for many luciferase enzymes. For this study, Renilla Luciferase interacted with coelenterazine to determine relative light output and kinetics [3].
+In the mechanism, coelenterazine reacts with oxygen to yield 1,2-dioxetane (compound 2). Subsequent loss of carbon dioxide leads to the intermediate, followed by emission of a photon.
 The image above is the chemiluminescent mechanism of coelenterazine. The bioluminescent mechanism is similar, except that the excited state molecule in bioluminescent is phenolate anion instead of the amide anion [2].