Our objective, as described in the Project Description and Project Motivation, is to construct a standard Biobrick assembly for the 5'UTR. We started our Literature Search in May to plan the design of our experiments. This has been documented and uploaded on the Wiki as well. The findings we incorporated in our project design have been accentuated here.
In 2018, Viegas et al. wished to understand the contribution of mRNA stability to heterologous protein production levels in E. coli Dh5α cell lines. They constructed their assembly following the rules of the Standard European Vector Architecture (SEVA) format. They used BBa_B0034 as their RBS and sFGFP as their markers to analyze UTRs constructed by them through rational design. The sfGFP gene was obtained from the BBa_I746908 plasmid. They used a constitutive promoter Prm following which is the 5’UTR. They conclude that the motifs they had inserted in the UTR construct of syn 2d and 2h were instrumental in enhancing gene expression. However, they also state that it would be better to test these constructs with a different combination of promoters and RBS. Based on the data of mRNA half-lives and gene expression analysis of the paper, we select the UTRs syn 2d, syn 2e and syn 2h for our experiments. These UTRs performed well as compared to the control by four to five folds, and had better stability. As suggested by the paper, we changed the RBS in our case to BBa_B0030. We also thought of using a GFP in our analysis. Professor Vito suggested that we could try changing the CDS as well. He also said that if we use the wtGFP, we would not be able to measure the decrease in fluorescence accurately. He insisted that we use truncated GFP as it has lower stability. In contrast, the complete GFP protein won’t be easily degraded, and we will get background signals from GFP produced long ago. Hence, we made the monomeric RFP BBa_E1010 our choice.
In 2020, Xiao et al. developed a portable 5’-UTR sequence for enhanced protein output in the industrial strain B. licheniformis DW2. This 5’-UTR could promote the accessibility of both the Shine-Dalgarno sequence and start codon leading to improved efficiency of translation initiation. The hairpin structure protected mRNA against 5’-exonucleases resulting in enhanced mRNA stability. They used the promoter P43 and observed that their 5’-element could effectively enhance the expression of eGFP by ~50-fold, and showed good adaptability for other target proteins including RFP, nattokinase and keratinase. They suggest that further work is needed in this area. We used the promoter BBa_J23100 and chose E. coli for our optimization process while they had chosen the Bacillus strains. They report that they could not correlate their data to the online RBS calculators. This further added purpose to our experimental validation and optimization processes for the 5’UTR.
In the meantime, we also had a meeting with Dr Joseph Wade. He recommended that we should test the naturally occurring 5’UTRs in E. coli as well. He explained that it would be great if we could attempt to understand the functioning of 5'UTR rather than only taking synthetic UTRs from other papers or 5'UTRs predicted by software. We conducted a literature search on the half-life data of the 5'UTRs and selected the naturally occurring 5'UTRs of E. coli with the highest mRNA half-life. We obtained these sequences from Ecocyc. We have discussed our discussion with Professor Wade on the Integrated Human Practices page.
Based on the suggestions of Dr Amanda Hughes, we had also set out to look for sequences with secondary structures that varied with the temperature that had a direct effect on translation (known as RNA thermometers) A primary investigation carried out on the sequence encoding the cold shock proteins concluded that the temperature sensitive nature of translation was not a result of the untranslated region, but of a series of codons in the open reading frame itself. Following this, we began a literature search on the sequence encoding the heat shock protein. In the untranslated region of the heat shock protein genes exists a sequence known as repression of heat shock expression (rpoH) that sequesters the ribosomal binding site and makes translation extremely difficult at lower temperatures, where the secondary structure is highly stable. However, at higher temperatures the secondary structure disintegrates and translation is significantly easier.
The literature search was accompanied by simulations carried out on NUPACK, which aided in the computation of the melting temperature of several sequences.
We also looked at cold shock proteins based on one of Dr. Amanda Hughes' suggestion. From the supplementary data of Bernstein et al in 2002, we found that cspF and csPH had high half-lives of 11.4 minutes and 6.2 minutes respectively. It was therefore concluded that trials would be carried out in the wet lab, where we would clone the 5'UTR corresponding to the cold shock protein gene in wild type E. Coli, upstream of our RFP and test them as well. We would then carry out regression to correlate experimental data with the predicted data from OSTIR and RBS Calculator. The date wise literature search analysis is presented on our Wiki Page as well.
We thank IISER Berhampur for their inputs in our Design stage.