20.109(F07): Transfection

Introduction

Luciferase is an enzyme that oxidizes its substrate, luciferin. The product, oxyluciferin, emits light in the blue range of the visible spectrum, 440-479 nm. The oxidation reaction is fundamentally different from another light emitting reaction you may be familiar with, fluorescence, as seen with GFP. During fluorescence absorbed light is re-emitted by the excited fluor, often toward the green range of the spectrum, ~505nm. Interestingly our luciferase source, Renilla reformis, has both a luciferase-luciferin pair as well as GFP. Consequently the oxidation reaction can lead to oxyluciferin luminescence and then to fluorescence, through energy transfer to GFP, giving the soft corals a blue-green glow.

Luciferin-luciferase pairs are widely used in nature for courtship, camouflage or baiting. Fireflies (also known as lightning bugs or more technically Photinus pyralis) use an ATP-requiring luciferin-luciferase pair and emit species-specific patterns of light as part of their mating ritual. Bioluminescent bacteria (such as Vibrio harveyi) can be found in symbiotic relationships with marine organisms. The fish give such bacteria a suitable home and in return the bacteria emit light to give the fish “night vision,” or to mask the fish’s shadow, effectively cloaking them from their prey. The usefulness of such luciferin/luciferase pairs was not lost to researchers who began isolating and sequencing luciferase genes from different organisms in order to clone them into useful vectors, such as the one we’ll use today.

Unexpectedly, there is little primary sequence similarity for luciferases from different organisms. This finding will be used to our advantage as we target mRNA from the Renilla luciferase gene for destruction while using the product of the firefly luciferase gene as an unaffected control. On the plasmid we will use, each gene is controlled by a strong constitutive promoter, leading to high expression levels of both luciferases when the plasmid enters a mammalian cell. Other plasmid features that you will be familiar with from experimental module 1 include an antibiotic resistance gene, in this case against the antibiotic ampicillin, that serves as a selectable marker in bacterial cells, a bacterial origin of replication, and multiple restriction sites that can be used for cloning.

In many ways, the assay for luciferase activity is a "standard" assay. The enzyme and substrate must react for a defined time and the amount of product gets recorded. What's a little unusual is that in this case the measured product is light and the light can be quantified using a luminometer. Today’s reactions are slightly more advanced than standard ones in that you’ll perform two sequential measurements. The first reaction is initiated with Beetle luciferin, a substrate for firefly luciferase. The light produced, called a “flash reaction” because it does not persist, will be measured for 10 seconds and then a reagent to stop the reaction will be added. The stop reagent also contains the substrate to initiate the second reaction. Coelenterazine reacts with Renilla luciferase in a “glow reaction” which decays more slowly but you will measure it for precisely 10 seconds so the lumens emitted can be compared to those from the firefly luciferase. There are several attractive aspects to these reactions including the assay’s low cost, high speed, great sensitivity (to attomolar amounts of luciferase, that’s 10-18th!) and wide dynamic range, linear through 7 orders of magnitude. It is also beneficial that there is no endogenous luciferase-luciferin pair in most experimental organisms.

There are two parts to today’s lab. Half the class will begin in the TC facility transfecting MES cells with the luciferase reporter plasmid and siRNAs. The other half of the class will begin by testing some control extracts from transfected cells to become familiar with the luciferase assay and analysis of the data that is generated. Midway through the lab period, the groups will switch places so everyone will have an opportunity to perform both protocols.