Software
Since the actual protocols state that the sRNA design must consider the first 24 nucleotides on the target mRNA without studying thermodynamic parameters, we had to design our first constructs following this methodology. In order to improve sRNA functionality we decided to create a python-based pipeline capable of designing sRNAs targeting an entire gene and then applying a scoring criteria following literature parameters. Since the mechanisms underlying sRNA.mediated gene regulations remain unclear to date, we have developed a neural network algorithm (trained with information of natural sRNA:mRNA pairs) in order to predict the regulation behavior (upregulation or downregulation) of the previously created sRNAs. It is important to note that since all of the created sRNAs have a Hfq-binding domain, the expected effect is downregulation, however since the effects of synthetic sRNAs have not been well characterized when the target regions are located far downstream, we decided to create the neural network model.
Image 1. Workflow diagram for design and improve of sRNA constructs for gene downregulation
Wet Lab
When developing our project we went through the Design→Build→Test→Learn cycle several times. As part of our original plan to be performed in the lab, we were considering using the M13KO7 helper phagemid to construct the non-modificated phages and test the silencing efficiency of the sRNAs in E. coli TG1. Nevertheless, Mexican suppliers of the M13KO7 did not have an available stock to be sold and to be arrived in a short period of time. We tried with an international supplier but a significant difference in price was notable, and we wanted to avoid all the shipping problems that would have been involved in this specific scenario, as we have been through with parts and reagents ordered by an international supplier. As part of the interaction with Ph.D. Alejandro Gonzalez, he offered us an aliquot of the VCSM13 helper phage that he had in our college laboratories readily available for us. VCSM13 helper phage is a derivative of M13KO7, and both are a good option when selecting a helper phage, nevertheless, before VCSM13 was offered to us M13KO7 was easier to access as is the one Mexican suppliers usually work with, but after Ph.D. Alejandro Gonzalez's offer, changing M13O7 for VCSM13 was an excellent option to optimize our two less available resources, time and money.
Our original plan also considered using a construct of the chimeric GP3 protein (BBa_4506000) and cloning it into PSB1A3 to perform the phage display in P. aeruginosa. Nevertheless, we presented some errors at the time our parts were evaluated by IDT due to its complexity to be synthetized, as a consequence we had to make some modifications to the GP3 chimeric protein for the phage display experiment. Ph.D. Alejandro Gonzalez helped us with this by kindly offering an aliquot of E. coli DH5α containing Pcomb3XTT backbone, which he used to successfully perform phage display in M13 bacteriophages, by changing also the N terminal of the P3 M13 protein. With this alternative we had to create a new derivative of the GP3 chimeric construct containing only the N terminal of the P3 protein of the PF1 phage, Pcomb3XTT had already the F1 replication origin and accessible cut sites to digest and then clone the insert, thus it was an excellent substitute to the original plan.
Image 2. Design of the cassette harboring the chimeric construct OmpA+ N term -Pfl
Image 3. Flowchart of the gBlock construction strategy for the synthesis of our chimeric protein