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 that we incorporated in our project design have been accentuated here.
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.
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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. <em>However, they also state that it would be better to test these constructs with a different combination of promoters and RBS.</em> 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 with respect 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. <ahref="{{ url_for('pages', page='human-practices3') }}">Professor Vito</a> 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.
