About our contributions
Since the reviewed literature states that the gene silencing through sRNA technology can be performed when the base pairing site is located far downstream of the start codon and the actual protocols for synthetic sRNA design rely upon the hybridization over the first 24 nts of the gene, our team decided to create a python pipeline for designing a dataset of sRNAs that can bind to downstream regions and then calculating the hybridization efficiency coupled with a sequence and folding score for then selecting the pairs with better punctuations.
As a result, we have improved the part Part: BBa_K1137010 (sRNA anti Cm) using the sequence for chloramphenicol acetyltransferase for designing a set of sRNAs that will base pair downstream the start codon. It is important to note that we have only changed the 24-nt space in which the sRNA baspairs with its target; the promoter, the Hfq binding domain and the terminator T1/TE remain the same.
Despite the fact that sRNA designers can create a dataset of sRNAs for the entire mRNA sequence, we have chosen only those with the higher scores inside 100 nts downstream the start codon, since most natural sRNAs base pair in this space.
As improvement, we present three more sRNA constructs: the (BBa_K4506004) with the higher score 1; (BBa_K4506006) with the higher score 2 and the BBa_K4506007 with the higher final score, which is a sum of the scores 1 and 2. The detailed mechanism for score calculation is on our Model and Software pages.
Figure 1. Scoring profiling of the created sRNA sequences (mRNA binding site + Hfq-binding domain) by our team and the one created by Paris-Bettencourt team (2013) for silencing chloramphenicol acetyl-transferase.
Since the design of sRNAs for targeting downstream regions over the CDS rather than the start of the gene can lead to higher hybridization efficiencies and higher sRNA structural features than the achieved with the conventional protocol, we consider that our software tool: sRNA Designer can be used by further teams in order to design better sRNA constructs and then characterizing the achieved silencing efficiency with lab experiments.