Difference between revisions of "Part:BBa K1732004"

Revision as of 00:26, 17 September 2015

pSB1C3-J23100-B0034-CDcel-Gaussia-His-B0015

J23100 constitutive promoter (BBa_J23100), a RBS (BBa_B0034), CDcel domain (BBa_K1732002), Gaussia luciferase (BBa_K1732003), and B0015 terminator (BBa_B0015). ______________________________________________________________________________________________________________________________

This DNA sequence, expressed from a constitutive, strong promoter allows for the Gaussia luciferase protein to be synthesized and was targeted extrecellularly into the media. The Gaussia luciferase proteins were reacted with the optimal concentration of coelenterazine to express luminescence, which was used to test the Carnegie Mellon DIY luminometer. The sequence was codon optimized for E.coli and contains a 6X His-tag for easier purification of the protein.

[Bioluminescent Mechanism of Coelenterazine

[2]

Coelenterazine is found in several aquatic organisms and is a luminescent substrate for many luciferase enzymes. For this study, PelB Gaussia 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 [2].

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 CD-cel Gaussia Luciferase, the location of the luciferase in the media was determined by comparing the light output of overnight grown culture of CD-cel cells with those of the pellet and the supernatant. The CD-cel Gaussia culture was spun down, and the pellet represented intracellular localization while the supernatant represented extracellular localization.

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

The graph shows the amount of light output produced from varying concentrations of coelenterazine when added to 100uL of CD-cel Gaussia cells grown overnight (5mL LB broth, 5uL Chloramphenicol, single cell colony). 10uL of coelenterazine was added and this volume stayed consistent. The overnight culture was diluted 1/10 with LB broth in order to measure the Klett OD and stay within accurate measurement range. The purpose of this is to see the effect of light output with increasing concentrations of coelenterazine. The goal was to find a concentration that plateau without maxing out the luminometer (TECAN). It was discovered that at a certain concentration, the light output stop increasing because the acidic methanol used to make the stock solution of coelenterazine began to interfere with the enzyme activity.

The two types of competent cells used were Mach and Top10 cells. Mach 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, but the decay rate is more significant . 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 and the jump in light output is temporary.

File:DONNA3.jpg

References

[1]Gaussia Luciferase [Fact sheet]. (n.d.). Retrieved September 14, 2015, from New England BioLabs Inc.

    website: https://www.neb.com/applications/cellular-analysis/reporter-systems/gaussia-luciferase

[2]Gonzalez, V. M., Jr. (2007). Synthesis, Luminescence, and Applications of Coelenterazine and its

    Analogs. University of Illinois Urbana-Champaign.

[3]Coelenterazine [Fact sheet]. (n.d.). Retrieved September 14, 2015, from Gold Biotechnology website:

    https://www.goldbio.com/product/1012/coelenterazine

Sequence and Features