In any scientific project there is a continuous cycle of making hypotheses, setting up experiments, measuring and evaluating the results. In this process the next set of hypotheses and experiments can therefore only be as good as the measurement techniques employed. Hence, high performing scientific measuring instruments have become the most costly investment many labs around the world choose to make. Fortunately the University of Groningen has an arsenal of excellent measuring equipment available spread around multiple buildings in the city that we were able to get access to.
Once impactful decisions have to be made based upon laboratory results, accurate measurement techniques become of immeasurable value. Nobody likes false positive or false negative results. Giving up on a promising line of experiments because of a mistake during measurement without ever knowing what could have been possible must be amongst the worst nightmares of the modern scientist. The Groningen iGEM team 2018 is happy to be free of such doubts because of the state of the art RP-HPLC UV-DAD, GC-MS, GC-FID, Nanodrop, Plate Reader machines, and many more that have provided us with reliable data to make the best decisions on how to proceed with our project and to get the best results towards our proof of concept. On this page we will highlight how some of these methods work, how we modified them and where their results come into our project.
What to measure? Styrene!
To prove our concept of a styrene producing, cellulolytic S. cerevisiae strain the detection of styrene is an essential part. For the identification and quantification of styrene, a variety of approaches that utilize different chemical characteristics of styrene are known. The purification procedure for styrene for measurement is different from the one for harvesting from a fermenter because the styrene only has to be detected and quantified, not purified for polymerization. To detect something in a mixture one has to find a signal that is both specific and sensitive for the compound of interest. This means that the generated signal cannot be created by something else that is present in the mixture at the point of measurement while the signal has to already be caused by small quantities of the analyte. Interesting physical and chemical properties of styrene that could be used to discriminate it from other compounds in the mixture include its UV activity at 245 nm, its high log P of 2,7, the high enthalpy in the reduced C-C and C-H bonds and last but not least its affinity to aromatic receptors in the human nose.
Which methods and detectors should we use?
To capitalize on styrene’s high log P, mainly chromatographic methods come to mind. These methods can identify styrene based upon its preference for a stationary phase over a mobile phase which leads to a difference in retention time. The signal for styrene detection can subsequently be generated in many ways, including UV absorbance measurement at 245 nm, measurement of an entire UV spectrum or even by burning the analyte as it comes off the column to detect forming carbonium ions.
How does chromatography work?
In chromatography, a liquid or gaseous mobile phase is lead over a stationary phase over a certain distance in a column. At the beginning of the column, a constant flow of new mobile phase is supplied. There is also a side for the injection of the sample for measurement. At the end of the column, a variety of signal generating and measuring devices can be installed that measure what comes off the column over time. Analytes that interact strongly with the stationary phase will reach the end of the column later. This so called retention behaviour is characteristic of the analytes physical chemical properties and reproducible under identical circumstances. Chromatographic techniques can be optimized in plenty of parameters: choice of stationary phase, choice of mobile phase, choice of signal generation, flow speed, temperature and usage of a gradient mobile phase. In a gradient mobile phase two or more solvents are employed whose composition changes over time. This allows for even better separation of peaks over time. To ensure reproducible measurements in chromatography, the machines have to be purged with fresh mobile phase after every run. To protect the column from contamination that might alter the retention behaviour of analytes, samples have to be freed of all enzymes, most cellular macromolecules and the lipid bilayer. The sample preparation for chromatography samples from living cells therefore commonly involves steps such as cell lysis, centrifugation, liquid-liquid extraction and filtration.
How did we employ RP-HPLC to detect styrene?
A Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method was evaluated for styrene identification by injection of styrene in ethyl acetate, measuring the retention time and recording a UV spectrum using a Diode Array Detector. Reverse Phase describes a chromatography method in which the stationary phase is apolar while the mobile phase is polar. The stationary phase of choice was a C18 column. The first mobile phase evaluated was a gradient system of water 100 % to 0 % and acetonitrile 0 % to 100 %. This was tested in a 60 minute run to determine at which percentage of acetonitrile the styrene would elute. As styrene eluted as late as 80 % acetonitrile it was decided to switch to a gradient mobile phase with water 100 % to 0 % and methanol 0 % to 100 %. In this case, styrene eluted at 50 % methanol. As nothing significant eluted at a low methanol percentage it was decided to validate and settle on a 60 minute run with a water 70 % to 0 % and methanol 30 % to 100 % gradient mobile phase. With this method, styrene eluted at 17,5 minutes.
How did we use GC-MS and GC-FID to quantify styrene?
Besides HPLC also Gas Chromatography (GC) was employed to measure styrene. Styrene as analyte is very suited for separation on a GC due to its volatile behaviour. A GC setup with a Mass Spectrometer (GC-MS) can be used for impeccable identification while GC coupled to a Flame Ionization Detector (GC-FID) is a very accurate technique for quantification. Because of the considerable sample preparation that is required to run samples in GC and the inevitable slight errors in these steps, a quantification of styrene is only possible if we can correct for the variance in the sample preparation. This is taken care of by addition of the internal standard 2-methylanisole to the liquid culture sample before any sample preparation steps are executed. The concentration of the standard before the sample preparation is known because the added volume of standard solution and the volume of sample are standardized. With the known standard concentration and volumes, the ratio of the area under the curves of the analyte peak and the standard peak is linearly proportional to the concentration of styrene in the sample before the sample preparation. For this to be possible and true however, the internal standard has to show almost identical physical chemical behaviour as styrene during the sample preparation while having a different retention time on the GC system.
The sample preparation for the GC-FID measurements started with mechanical cell lysis, liquid-liquid extraction using benzene and centrifugation. 2-methylanisole behaves very similarly to styrene during this extraction protocol as it has the same log P while also having roughly the same size of conjugated ring system for stacking interactions with the benzene extraction solvent.
The Flame Ionization Detector utilizes the fact that fast combustion of carbohydrates leads to the formation of carbonium ions at a constant rate linearly proportional to the amount of carbon atoms inside the analyte. The impact of these positively charged carbonium ions like CHO+ on a negatively charged metal surface can be measured as a change in electrical current in the plate. When plotted into a chromatogram, the response ratio of the analyte and the internal standard electrical current signal over time (AUC) can be used to calibrate the amount of analyte in the sample before sample preparation using a calibration curve.
Introduction to Nuclear magnetic resonance spectroscopy
NMR (Nuclear Magnetic Resonance) spectroscopy is an extremely powerful analytical technique frequently used in chemistry to determine the content and purity of a sample. The technique relies on the specific magnetic properties of the nucleus of each atom in a molecule.
Each nucleus can be thought of as a ‘tiny magnet’, because these spinning charged particles create a small magnetic field with random orientation. When a magnetic field (Bo) is applied it will result in two energy levels with specific orientation; in alignment (low energetic level) or in opposite alignment (higher energetic level) with the applied magnetic field. When the nucleus subsequently relaxes back into the original orientation it will send a signal (chemical shift) which is measured by a sensor.
Phosphorus NMR spectroscopyThe distinct NMR techniques 13C-, 19F- and 31P-NMR depend on the specific excitation frequency of each atom. We chose 31p-NMR spectroscopy as an indication for the phosphorylation reaction.
The spectrophotometer: a measurement workhorse
One of the most important measurement devices at our teams disposal is the Implen Nanophotometer spectrophotometer. This is an incredibly versatile measuring device and requires only 0.3 µl of sample. Our team used the Nanophotometer to measure DNA concentrations after minipreps, when setting up PCR reactions, and when preparing samples to send out for sequencing. Without these sensitive measurements, the amount of DNA used for PCRs and transformations of strains would be determined a lot more arbitrarily, and the preparation of sequencing samples would become almost impossible given the sample concentration requirements.
The Nanophotometer is furthermore used to measure the optical density of cultures, as it is accurate over a broad range of OD’s. No dilutions have to be made, as opposed to most cuvette-based systems. This is due to a path length of only 0.67 mm , instead of 10 mm, and this reduced path length equals a 15 times dilution compared to a cuvette based system.
Other important features of this measurement device are the capability to measure protein concentrations as well and to perform kinetic analysis over time. These features allow the Nanophotometer to perform enzyme activity analysis. Equal amounts of enzyme can be loaded between samples, and if the enzyme substrate and product show different absorbance peaks, the conversion over time can be determined.
The plate reader: providing growth curves of strains of interest.
Plate readers are measuring devices that accept samples in 96 well plates, and can measure all wells at a high speed. The device can measure at multiple timepoints, has a temperature control function, and can vortex the sample plate during the incubation steps. Depending on the device, multiple variables can be measured, for example the optical density at multiple wavelengths, fluorescence, and absorbance. These factors combined make it the obvious choice for screens of multiple samples, kinetic measurements, growth curves, et cetera.
Because of the aforementioned properties of the plate reader, it is the perfect tool to make growth curves of our different Saccharomyces cerevisiae strains on cellobiose and cellulose. Samples are prepared and loaded into a 96-well plate. The OD600 is measured in each well at multiple time points over a 24 or 48 hour period, depending on the observed growth rate.
- Percival Zhang, Y. H., Cui, J., Lynd, L. R. and Kuang, L. R. A transition from Cellulose Swelling to Cellulose Dissolution by o-Phosphoric Acid: Evidence from Enzymatic Hydrolysis and Supramolecular Structure. Biomacromolecules, 7, 644-648 (2006)
- Implen. Implen N50/N60 tech specs. https://www.implen.de/product-page/implen-nanophotometer-n60-microvolume-spectrophotometer/tech-specs/