Microfluidic platform
- We designed and manufactured a device capable of multiplexing reactions for the detection of antibiotics by producing Bacillus subtilis culture droplets, and simulating the conditions of biosensors activation in droplets with the use of antibiotics.
- We used microfluidic technologies, which facilitates the controlled and high-throughput generation of stable and monodisperse droplets using biologically relevant contents.
- In order to determine the best conditions for the generation of droplets in a specific geometry, a numerical analysis was performed based on the current physical models for droplet generation and breakup.
- Based on the numerical analysis, boundary conditions were established for the flow rates in a T-junction for the creation of droplets for both the continuous and dispersed phases.
- Such conditions were tested in a computational fluid dynamics (CFD) simulation to validate the design and conditions in a time-dependent study.
- Droplets were successfully generated in such simulations, but certain flow rates were found to be a more efficient combination for droplet generation.
- A microfluidic device with the proposed geometry was fabricated using standard soft lithography techniques.
- The polydimethylsiloxane (PDMS) devices were bonded onto a glass slide covered with PDMS and sealed to form the microfluidic channels using a plasma surface activation.
- The proposed conditions were tested in a microfluidic device and results were compared with the simulations.
Whole cells biosensors
- In this module we demonstrated that the plasmid PBS1C3 was successfully ligated to a promoter+RGT and transformed for the 3A assembly strategy in E. coli DH5 alpha
- The DNA extracted from the white colonies in each agar plate was digested with the corresponding restriction enzymes and an electrophoresis gel was run.
- We simulated an agarose gel in the Snapgene program to visualize the band sizes of our parts and compare them with our gel. The size of the plasmid band was between five and six kb, while the ligated promoters were around the the 1.5 kb band.
- We confirmed that promoter ypuA was transformed in the following images:
Amplification cascade
- We ligated the promoters with the cascade amplifying circuit through Golden Gate cloning (go to protocols to see what we used), and transformed the products in E. coli DH5 alpha, performing also a blue-white screening.
- Afterwards we ran an electrophoresis gel to further confirm that the ligations were successful. At the same time we made a simulation of the same gel in Snapgene
Mucolytic
- We transformed E. Coli with the different constructs of this module and noticed that both the bacteria transformed with the sfGFP and that with the chimeric protein showed to be highly expressed according to their phenotypes.
- To see if this was due to a leakage, we inoculated them in 200μM IPTG lb agar plates.
- Plates with IPTG didn’t showed to be fluorescent, so we decided to perform a fluorescence curve assay in a microtiter plate.
- We observed a strong leakage, in the absence of IPTG but the behaviour looked like that for a normal induction.
- To quantify the amount of protein present in the eluted proteins, we did a Bradford assay.
- The following figures show the results from the assay. Including the calibration curve and the values used to calculate the linear equation. The results from the measurement of our sample are also presented; it is evident that the less protein present is the chimeric sialidase-sfGFP protein, this might be because of the size of the protein it is harder to secret.
- We ran a gel with both the supernatants and cell lysates and their different fractions of elution.
Fluorescence vs time of sfGFP production induced at 0, 40, 200 and 400μM.
SDS-PAGE from left to right the samples are, the lysate of an induced E. Coli producing the sfGFP, 20mM Imidazol fraction of Ni=NTA purification, sfGFP 150mM Imidazol elution fraction, supernatant from sfGFP producer, negative control (an E. Coli transformed with an Psb1c3 only containing a promoter between the prefix and suffix), sialidase induced supernatant and non induced supernatant, and a sfGFP non induced. Induction was done at 400μM.