Disclaimer: While out project may not be using luciferase, its reactions still follow the same processes as those of our own lux gene, making it imperative for anyone working in this field (and in specificity our project) to have a basic understanding of this topic.
In regards to the enzyme luciferase, a common and necessary component in the bioluminescence of many organism (i.e. fireflies), there are multiple steps and chemicals which are utilized in the reactions to produce the bioluminescence in the organisms. It should also be noted the usage of energy during bioluminescence for any organism is high (6 molecules of ATP for a single photon of light, while assuming 100% efficiency within the reaction). This makes the prolonged usage of bioluminescence extremely taxing for any organism, requiring a large amount of nutrients for the process to be carried out.
Taken From Text:
"Currently, it is known that the blue-green light emission of bioluminescence, such as that produced by the bacteria Photobacterium phosphoreum and Vibrio harvey, arises from the reaction of molecular oxygen with FMNH2 and a long-chain aldehyde to give FMN, water and a corresponding fatty acid. The luciferase enzyme catalyzes a mixed function oxidation of the long-chain aldehyde and FMNH2. The reaction is highly specific for FMNH2, which is protected against autoxidation once bound to the enzyme. The bioluminescent reaction is as follows:”
FMN- FMN is the most common form of the (vitamin B2) (riboflavin) which is found in cells and tissues. It takes more energy to produce than other forms of (riboflavin) (non-phosphorylated form), but is much more soluble in water. FMN is produced from riboflavin by the enzyme riboflavin kinase, and functions as prosthetic group {the nonprotein acid constituent of a conjugate protein, as the heme group of hemoglobin.} of various oxidoreductases {an enzyme that catalyzes the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor. } including NADH dehydrogenase.
Chemical Formula: C17H21N4O9P
FMNH2- The reduced form of FMN.
The reaction following the processes mentioned above is:
FMNH2 + O2 + R-CHO à FMN + H2O + R-COOH + Light (~ 495 nm)
with the reaction being highly specific for FMNH2.
The energy produced from this reaction is enough to produce light (bioluminescence) {amount of energy needed for the production of light is 60 Kcal mol-1}. Because of the large energy requirements mentioned earlier (6 ATP per photon), this process is reserved for when the bioluminescence of the organism is physiologically necessary. This creates a predicament for any prolonged bioluminescence within most organisms (encompassing our experiment as well) in regards to the large nutrient requirements, making our idea of a synthetic-bioluminescent light source extremely expensive (in terms of energy) and un-cost effective.
Another form of bioluminescence regulation is known as auto-induction which is the cell-to-cell communication, tying gene expression to bacterial cell density (this was first discovered in Vibrio fischeri).
Taken From Text:
Quorum sensing involves the self-production of a diffusible pheromone called an autoinducer (AI), which serves as an extracellular signal molecule that accumulates in the medium and evokes a characteristic response from cells (42).
Dilute 400μl of overnight liquid culture into 20ml of broth
Incubate at 37⁰C, shaking at 225rpm for 90 minutes
Spin down for 2 minutes at 7000rpm at 4⁰C
Discard supernatant, resuspend pellet in 10ml of 50 mM CaCl2, keep on ice
Repeat centrifugation for 2 minutes at 7000rpm at 4⁰C
Discard supernatant and the resuspend pellet in 1ml of 50 mM CaCl2, keep on ice
CaCl2 competent cells can be kept on ice in the fridge overnight
Transformation of CaCl2 Competent Cells
1μl of plasmid DNA was added to 100μl of competent cells
Samples were incubated on ice for 20 minutes
Heat shock was cried out at 37⁰C for 5 minutes
Cells were kept on ice for 2 minutes
200μl of broth was added and the cells incubated at 37⁰C for expression step (time varies dependent on antibiotic resistance gene in the plasmid that has just been transformed into the cells):
100-200μl of transformed cells was spread on dried L-agar plates with required antibiotic(s) to select for plasmid(s)
Chloramphenicol resistance = 90 minutes
Kanamycin resistance = 60 minutes
Ampicillin resistance = 30 minutes
CaCl2 competent cells can be kept on ice in the fridge overnight
