Team:Nevada/Project/Assay

Introduction

In light of the growing energy crisis, much research has been devoted to finding economical means of producing renewable fuels. Traditional methods for obtaining biofuels have relied mainly on the fermentation of agricultural crops. However, there are a number of problems with this approach: the reduction in land available for food production, relatively low levels of CO2 biofixation, and large biomass requirements. Our project aims to overcome these problems by utilizing E. coli for the production of biodiesel (C-12 fatty acids) and bioethanol. In the past there have been a number of examples of biofuel production in E. coli; however 30-40% of production cost is based on media costs (Galbe et al., 2007). Our project will surmount these high production costs by engineering the cyanobacteria, Synechocystis PCC 6803, to secrete large quantities of glucose that will feed our biofuel-producing E. coli. Cyanobacteria and E. coli will be co-cultivated in an apparatus that allows for the mutual transfer of carbon to produce biofuels. Not only will this project provide an efficient means for producing biofuels without the need for a carbon source, but it will also create a novel cooperative system between bacterial species that may have further industrial implications.

Throughout the duration of this project, enzymatic assays were used to confirm the function of the genes used to transform E. coli and quantify the secretion of biofuel products. A hexokinase assay was used to measure glucose/fructose secretions from cyanobacteria, and oxidase/peroxidase assays were used to quantify free fatty acid and ethanol production from E. coli.

Assay Development

Most techniques for directly determining the concentrations of sugars, alcohols and fatty acids in culture media involve multiple steps, and may not distinguish between, for example, different types of sugars. We overcame these problems by using enzyme assays which, upon the addition of the sugars, alcohols, or fatty acids, result in the formation of other compounds which can be measured directly.
For all assays, samples of Synechocystis or E.coli medium were taken, and centrifuged to remove particulates. These samples were then added directly to assay mixtures containing enzymes and additional substrates necessary for the formation of compounds whose concentrations we could measure directly.
Coupled enzyme assays for Glucose, Fructose and Sucrose uses enzymes to catalyze a series of reactions, resulting in the formation of NADH, which we can measure directly in the spectrophotometer.

Assay Description: Invertase enzyme will be directly added to sample media to split sucrose into D-glucose and D-fructose, which are then added to a Hexokinase/Glucose-6-phosphate DeH assay mix, which will produce one NADH molecule for every one glucose molecule added. NADH can be measure on the spectrophotometer at 340 nm and can be quantitated using Beer’s law and the NADH extinction coefficient (shown above). Because the assay is glucose specific, the first reading will quantitate glucose present, then a Phosphoglucose isomerase enzyme will be added to the assay mix to convert fructose-6-phosphate into glucose-6-phosphate and a second reading will be taken, the increase in absorbance will be used to quantitate fructose present.
<img src"">

*It should be noted that fructose is produced naturally by wild-type Synechocystis. It was therefore necessary to measure constitutive fructose production in wild-type cultures.

Fatty Acid Production was measured using the EnzyChrom Free Fatty Acid Assay Kit from Bioassay Systems according to the manufacture’s protocol. This kit uses a three step assay.
Assay Description: Fatty acids are enzymatically converted to acyl CoA and then to peroxide. A Peroxidase then uses the resulting peroxide to oxidize a dye substrate forming a pink colored product with optical density (O.D.) at 570 nm.

<img src="">

Ethanol Production

Ethanol Production was measured using a similar protocol to the free fatty acid assay:

<img src="">
Assay Description: Alcohol Oxidase converts primary alcohols like ethanol and diatomic oxygen into a formaldehyde and a peroxide, respectively. The peroxide is then converted into two molecules of water by a peroxidase using an ABTS substrate as an electron donor. The resulting oxidized ABTS will absorb at 405nm. There is a 1:1 ration of ethanol to oxidized ABTS molecules; therefore we can use the molar extinction coefficient of oxidized ABTS in order to quantitate the amount of ethanol originally present.

The fatty acid and ethanol assays were quantitated by generating standard curves of absorbance vs substrate concentration using known quantities of palmitic acid or ethanol standards.

Assay Description: Alcohol Oxidase converts primary alcohols like ethanol and diatomic oxygen into a formaldehyde and a peroxide, respectively. The peroxide is then converted into two molecules of water by a peroxidase using an ABTS substrate as an electron donor. The resulting oxidized ABTS will absorb at 405nm. There is a 1:1 ration of ethanol to oxidized ABTS molecules; therefore we can use the molar extinction coefficient of oxidized ABTS in order to quantitate the amount of ethanol originally present.

Assay mix components:

1. Alcohol Oxidase Enzyme (A.O.) 2. Peroxidase Enzyme (POD) 3. ABTS (Azino-bis-(3-Ethylbenzothiazo line-6-Sulfonic Acid) substrate

Assay chemistry: Two step coupled assay

1. Ethanol + O2 →A.O.→formaldehyde + H2O2 2. H2O2 + ABTS→POD→2H2O + Oxidized ABTS

Sample Calculation:

A = є•c•l

Є=ABTS millimolar extinction coefficient = 36.8 L/mMol•cm

A=0.654

c = A/є•l = 0.654/(36.8 L/mMol•cm)(1cm)= 17.77 µM

Quantification of Fatty Acid Secretion

Fatty acid secretion was determined using the EnzyChrom Free Fatty Acid Assay Kit from Bioassay Systems according to the manufacture’s protocol.

Assay Description:This kit uses as one step assay in which fatty acids are enzymatically converted to acyl CoA and then to peroxide. The resulting peroxide reacts with a dye to form a pink colored product with O.D. at 570 nm. There is no extinction coefficient for this colored product a standard curve my be created in order to obtain a linear equation that can be used to determine unknown concentrations.

For further assay information view the Bioassay Systems’ Free Fatty Acid Assay Kit manual.

Standards:

Palmitic Acid standards of the following concentrations: 1000µM, 600µM, 450µM, 300µM, 200µM, 100µM, and a blank standard (no palmitic acid). These standard were used to create a standard curve by plotting [Palmitic Acid] against ∆A @ 570nm (∆A=standard absorbance – blank absorbance, or background). The standard curve was then used to give a linear equation of Y=mx+b. This equation can then be used to determine unknown sample concentrations by plugging the absorbance of the unknown in for Y and solving for x.