Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors

This method can help answer several key questions involved in the production of human milk oligosaccharides. First, how much of the product do I have? And second, what is the specific carbohydrate linkage?

The goal here is to simplify and speed up the process of quantifying and characterizing these human milk oligosaccharides. This method has two advantages over the current state of the art. First, it's amenable to high-throughput screening.

So, you can imagine testing thousands of different conditions or perhaps even a library of synthesis enzymes in one day using this technique. Also, it uses the unique substrate specificity that nature has evolved in enzymes to give us information about the carbohydrate linkage of those oligosaccharides. We believe that this method can improve the production of human milk oligosaccharides, which in turn can lead to better quality infant formula that more closely mimics human breast milk and help close the gap between breast-fed and formula-fed babies.

User discretion is recommended when deciding what method to use for lactose removal based on cost, throughput and equipment availability. Care should be taken to ensure optimal lactose removal. The day before the experiment, seed 5 milliliters of LB medium supplemented with kanamycin and carbenicillin from a fresh bacterial colony using sterile techniques.

Incubate all cultures overnight at 37 degrees Celsius with agitation. The next day, transfer 50 microliters of an overnight culture into five milliliters of fresh LB M9 media with kanamycin and carbenicillin. Incubate at 37 degrees Celsius with continuous shaking until the culture has reached an OD600 between zero point five and zero point seven.

First, transfer the overnight cultures into one point five milliliter tubes and centrifuge at 12, 500 times g for one minute. Discard the supernatant and re-suspend the pellet in 500 microliters of 1x PBS to prepare a single cell suspension. Transfer the single cell suspension to fax tubes.

Use a flow cytometer to excite GFP and collect FITC fluorescence levels as outlined in the text protocol. To make a calibration curve, make eight to 10 dilutions of the standard 2'FL in the range between zero and 2500 milligrams per liter. Culture the 2'FL bio-sensing cells and induce them with the standard dilutions as outlined in the text protocol.

Start a culture of a 2'FL producing strain in five milliliters of LB media and grow it overnight at 37 degrees Celsius. The next day, subculture at 1%in 250 milliliters of prepared minimal media and add glycerol to a final concentration of 10 grams per liter. Incubate at 37 degrees Celsius until the culture reaches and OD600 between zero point five and zero point seven.

Then add IPTG to a final concentration of zero point five millimolar and lactose to a final concentration of two grams per liter. Incubate at 37 degrees Celsius with continuous shaking for 24 hours. The next day, transfer the overnight culture to sterile 50 milliliter tubes and centrifuge at 900 times g for 15 minutes.

Discard the supernatant and re-suspend the pellet in deionized water. Repeat this wash three times to remove any residual lactose. After this, re-suspend the pellet in five milliliters of deionized water.

Place the cells on ice and use a sonicator at 30%power in 30 second pulses to lyse the cell suspension. Centrifuge the lysed cells at 2, 000 times g for 25 minutes. Remove the supernatant and pass it through a sterile filter.

Store the filtered supernatant at four degrees Celsius until ready to analyze. Next, inoculate a culture of 2'FL bio-sensing cells. At the mid-log phase, induce the cells with the cell lysate at concentrations equivalent to that of those 2'FL used to make the calibration curve.

Perform the calibration as outlined in the text protocol. After this, run the samples on a flow cytometer. Generate a dose response curve by plotting the fluorescence output against the oligosaccharide concentration and compare the response of the standards to the cell lysate.

First, dissolve FL alphalyzed beta galactosidase to 1, 000 units per milliliter in one millimolar magnesium chloride. To determine the optimum concentration of enzyme needed, grow an inoculum of 2'FL bio-sensing cells and at the mid-log phase, induce with lactose and 2'FL. To 100 microliter cultures, add variable amounts of beta galactosidase up to 12 units.

Let the cultures grow overnight at 37 degrees Celsius with continuous shaking. Measure the fluorescence as described in the text protocol and calculate the optimum units of enzyme needed by determining the minimum enzyme concentration to achieve the desired attenuation of signal. To 2'FL producing cells grown for 24 hours add the optimum units of beta galactosidase and incubate overnight at 37 degrees Celsius, determine the 2'FL titre as previously described.

First, start a 25 milliliter culture of a 2'FL producing strain. Once the strain is induced at the mid-log phase, incubate the culture at 37 degrees Celsius for 24 hours. Next, inoculate an E.coli BL21 culture in LB media.

Grow it to mid-log phase at 37 degrees Celsius then add 15 milliliters of this culture to the 24 hour 2'FL culture. Incubate at 37 degrees Celsius for three hours. Then, determine the 2'FL titre as previously described.

To begin, start a 25 milliliter culture of a 2'FL producing strain. Once the strain is induced at the mid-log phase, incubate the culture at 37 degrees celsius for 24 hours. Add two volumes of 100%ethanol to the culture and shake well.

Incubate at 37 degrees Celsius for four hours. Then, centrifuge the suspension at 900 times g for 15 minutes. Collect the supernatant and incubate overnight at four degrees Celsius to precipitate the lactose.

Pass the solution through filter paper to remove the precipitated lactose. Collect the filtrate which contains the cell lysate and determine the 2'FL titre as previously described. In this study, a high-throughput strategy is used for the linkage specific detection of fucosylated human milk oligosaccharides.

This is accomplished by building whole cell bio-sensors by genetically engineering E.Coli which, upon induction with specific glycans, respond with a fluorescent signal. The fluorescent signal under different conditions of induction as analyzed on a flow cytometer, shows over a 100 fold increase in fluorescence with the 2'FL bio-sensor compared to the vector only control or the bio-sensor for 3'FL. This shows the high linkage specificity of the bio-sensor.

The sensitivity of the assay can be determined by generating a dose response curve with varying carbohydrate levels. The dynamic linear range of 2'FL detection, is seen to be between 40 and 400 milligrams per liter and the limit of detection is four milligrams per liter. This assay can be carried out over the course of a working day as seen in the snap shot measurements of the dynamics of reporter expression.

The bio-sensing cells can be used to detect and quantify 2'FL from an engineered 2'FL producer strain using a number of strategies to reduce signal from exogenous lactose so that the signal read out would directly correspond to the 2'FL concentration. One strategy is to enzymatically degrade the lactose using a beta galactosidase that does not act on modified lactose. Another strategy utilized ethanol which not only selectively precipitates lactose but also lyses the producer cells since cells lysis is a necessary step down stream.

Finally, the most effective but time consuming method is to pellet and wash the cells prior to cell lysis to release the intracellular 2'FL. Once we establish this method, we were able to expand the repertoire of oligosaccharide targets using different enzymes. Currently, we can detect nine different oligosaccharides and we're working on more.

While optimizing this assay, we noticed the robustness of our enzymes allowed us to come up with a technique that gives a linkage specific read out in about five minutes. Although none of the reagents used here are particularly hazardous, those using this method should follow standard biohazard and biocontainment procedures including wearing proper PPE.