We have created a library of 5'UTRs that gives a range of expressions. The key learning was how just nine nucleotides were enough to influence the gene expression, as observed in the case of cspF. We are still trying to interpret the complex structures of the naturally occurring UTRs. Our next experimental design shall be more complex based on our understanding and observations of GC content. We further plan to add a complexity factor to our next set of experiments by looking at RNAse cleavage sites. We have realised that experimental results are quite different from software predictions. This further justifies the need for more work and attention in the area of 5'UTR.
We conducted our experiments in two types of strains: a cloning strain (DH5alpha) and an expression strain (BL21(DE3)), and obtained expression values with a Spearman's rank correlation coefficient of 0.87. Such a high value indicates that the overall expression trend remains the same across these two strains. We also wish to conduct these experiments in wild-type strains like MG1655 and other types of strains so that the data we generate and contribute to the Library is expansive and useful to as many researchers as possible. We further wish to develop correlations based on experimentally validated data and build on our 5'UTR Library. We have completed the second engineering cycle and are now contemplating the design of the third, as described on our engineering cycle pages.
