Electronic device

We were able to express the protein with which we pretend to immobilize endocrine disruptive compounds (EDCs) in a Quartz Crystal Microbalance (QCM) in order to build a biosensor that quantifies these compounds.

To measure E0DCs in a flow rate, an immobilized hERa gel would be placed on the quartz crystal microbalance (QCM), and a  piezoelectric; it will relate differences in electrical potential and applied mechanical loads. An electronic circuit was developed to control the frequency of an alternating current applied between the crystal terminals and achieve resonance. When a small mass, on the order of micrograms, is placed on the surface, the difference in frequency increases and a calibration curve is constructed to determine the weight placed.

The first measurement would be the clean water control sample and then following with unknown concentrations of the compound to be analyzed. This is for the mathematical model to predict the amount of mass that has bound to the gel.
 

Biosensor model prototype

Although we didn’t get to join both the biotechnology and the mechatronics contributions by immobilizing ESR1 in the QCM, our project has established the beginning of a solution to the lack of normativity regarding endocrine disrupting compounds in such an important resource as water. It has proposed a friendly, small and resistant device to study these compounds in water. It has set a precedent to keep studying and characterizing these compounds, to eventually diminish or eliminate their effects on not just human health.

Degradation of EDCs through a laccase

We were able to verify the enzymatic activity of our laccase through a colorimetric assay and we were also capable of purifying our laccase through the development of a standardized protocol (Nickel affinity).
Our colorimetric enzymatic assays demonstrated that our laccase is functional and that laccase expressed at the culture soluble fraction has a greater enzymatic activity than laccase expressed in culture medium.

This was demonstrated by measuring the absorbance of our laccase samples with the colorants once a day, and producing degradation percentage curves of each colorant with each sample. Degradation by pH was tested and discarded by measuring pH after the assay, which did not exhibit any changes.

Also, we were successful at the purification through Nickel affinity chromatography of our laccase, which was verified through a SDS-PAGE, where the immobilized in Nickel resin sample exhibited enrichment, as shown below.

Next, we carried out a laccase-catalyzed degradation of BPA assay by measuring the absorbance at 290 nm. The reaction mixture contained BPA working solution, citrate buffer and the purified laccase from Trametes versicolor. Reactions were  incubated at 35°C and 250 rpm for different times. The negative control consisted of inserting citrate buffer to the reaction mixture instead of laccase. From these absorbance measurements, the following graph was obtained, proving that in the presence of laccase, BPA is degraded.
These discoveries can lead to the implementation of a bigger scale purification system for a huge laccase production that can help to degrade endocrine disruptive compounds massively.  This takes us a step closer to the addition of a unit operation for residual water treatments that will give us cleaner, healthier water. 
For laccase degradation assay, our next step would be to try our purified laccase and other EDCs other than BPA, because we still need to obtain more accurate results by further testing at different conditions, concentrations, temperatures, and laccases.

To read more information about these results, please check the information here. 

Novel Selection Marker

We demonstrated that culture medium solutions with glycerol inhibit microbial growth. We prepared LB culture medium with 0, 5, 10, 15 and 20% glycerol (v/v). We observed how in solid culture media there were less colonies in greater glycerol concentrations, and we measured optical density at 600 nm for liquid culture media periodically, for 12 hours. From those measurements, we plotted microbial growth curves, to note that as glycerol concentration rises, the optical density after 12 hours of the culture decreases, and the slope of the exponential phase flattens. The microbial growth curves are shown below.

We demonstrated that culture medium solutions with glycerol inhibit microbial growth. We prepared LB culture medium with 0, 5, 10, 15 and 20% glycerol (v/v). We observed how in solid culture media there were less colonies in greater glycerol concentrations, and we measured optical density at 600 nm for liquid culture media periodically, for 12 hours. From those measurements, we plotted microbial growth curves, to note that as glycerol concentration rises, the optical density after 12 hours of the culture decreases, and the slope of the exponential phase flattens. The microbial growth curves are shown below.​

This proves that glycerol can be used as a selection marker for synthetic biology and genetic engineering protocols, and represents a safer alternative because it avoids the creation of super resistant bacteria. However, for it to be a useful selection marker, we need something for it to allow microbial growth.

Keeping this in mind, we used genetic engineering to perform a gene knockout protocol to DH5a to introduce a genetic cassette that contains catalase and allows bacteria to degrade hydrogen peroxide (product of the reaction of aldO, a gene we introduced) produced in response to glycerol, giving them the ability to grow in a culture medium with glycerol. 
Even if we didn’t get the results we expected, we opened up a very valuable opportunity to implement safer and cheaper laboratory practices all around the world.