Wet Lab experiments
As part of our original plan, we designed all of our parts flanked with a set of M13 primers in order to have more DNA without the necessity of cloning in bacteria, a pair of AXTL primers (designed by us) to perform colony PCR and confirm a successful cloning procedure when needed. We have successfully amplified by PCR all of our synthesized parts by IDT (gBlocks containing sRNA’s, chimeric GP3 protein). However some of our parts were synthesized by Twist, and we noticed that we could amplify the parts when the DNA template was from the resuspended stock, but we could not use the PCR product as a new template since no amplification was obtained from this, even when changing the primers, polymerase and reaction conditions (annealing temperature). This last problem was mainly attributed to the bad shipping conditions that caused the 96 well plate containing our parts to arrive open and to be held at room temperature for almost two months.
We first planned to test our silencing antibiotic resistance gene system in resistant E. coli TG1 strain. We knew from the very beginning that we could not work with acquired antibiotic resistant P. aeruginosa due to biosafety reasons, so we only planned to test the phage display in this microorganism. To test the silencing efficiency of our sRNAs, we designed some sRNAs to silence ampicillin and chloramphenicol resistance in E. coli TG1, this strain would be transformed with PSB1A3 or PSB1C3 plasmids, so it can acquire ampicillin or chloramphenicol resistance respectively depending on the case. The E. coli DH5α strain would be used to be co-transformed with the auxiliar phagemid VCSM13, containing all the components of the M13 bacteriophages, and with the plasmid containing the sRNA (PSB1A3 or PSB1C3) that would silence the pertinent resistance gene.
Figure 1.
Just before we sent our parts to be synthesized, we also designed some sRNAs to silence RFP, so we can infect with the M13 phages the E. coli TG1 cultures containing RFP and quantitatively measure by fluorescence our rate of infection. Referring to the phage display, it has been already said that we were not planning to work with resistant induced P. aeruginosa. This phage display consisted in the construction of a chimeric GP3 protein, by changing the N terminal of the M13 P3 protein, which is the main implicated protein in the process of attachment with the host cell, by then P3 protein of the PF1 phage.
As mentioned in the notebook we presented several issues in all our cloning attempts, always learning from the mistakes and creating new strategies to get better results. A lot of time was invested in getting a good amount and quality of digested backbone, so as a consequence we had to focus our attention on working with a special selection of our sRNAs. This selection consisted of working only with the manual designed sRNA, the final sRNA which was the one that contemplated both of the models our software is based on, and if available, due to the degraded parts, the S2 model (Vazquez-Anderson et al. model). For the cloning we were using the traditional method, the parts were digested with EcoRI-PstI and EcoRI-SpeI in relation to the corresponding site cuts that were added in the prefix and suffix of each gBlock. After three cloning attempts and using a molar ratio of approximately 7:1 in each ligation reaction, we got some colonies with PSB1A3+S2 CAM and PSB1A3+Manual CAM. We proceed with the corresponding colony PCR using AXTL primers to amplify the insert and the outcome was positive.
From left to right: Control, colony 1 from PSB1A3+S2 CAM, colony 2 from PSB1A3+S2 CAM, colony 1 from PSB1A3+Manual CAM., colony 2 from PSB1A3+Manual CAM.
Figure 2.
Microfluidic Encapsulation Simulations
The proof of concept related to the microfluidic encapsulation of the bacteriophages is based on the simulation of the microfluidic coaxial device described in Design, in this sense, the simulation shows the liposome-phage complex, projecting the injection of the organic phase into an aqueous phase using a constant and controlled flow rate to induce a laminar flow. Our modification relies on the use of this technology for the encapsulation of M13 phages since this type of device has already been used for similar purposes [2].
Figure 3.
Since we were not able to synthesize this microfluidic device, we used COMSOL 6.0 to simulate the particle tracing using fluidic dynamics, this allowed us to obtain velocity fields that tracks the trajectory of the phages and provides us insight of the viability of the encapsulation. to learn more about the results of our simulations see Results.
Nebulizer equipment development
In order to simulate the nebulization of the liposome-phage complex our team develop as proof of concept a ultrasonic nebulizer, that approximates the nebulization of the phages in our design, this nebulizer constitutes a prototype of the final product and aids as a practical help to understand how the final product might work.
Figure 4.
Into this prototype we introduced the necessary electronics for the evaporation of the liquid contained into the superior chamber, the designing of the nebulizer was done using solid works and the 3D impression 3D was done using SLA/DLP.
Software proof of concept
Since our pipeline: sRNA Designer can perform sRNA creation for other bacterial genes in addition to those related to antibiotic resistance, we have compared the sRNAs designed by Cho & Lee (2016) in their study for silencing adhE1 gene expression in Clostridium acetobutylicum following the rules of Paris-Bettencourt (2013) iGEM team.
Figure 5.