Difference between revisions of "Part:BBa K4511006"

Latest revision as of 14:49, 12 October 2022

dtRNA1v2

Degradation-tuning RNAs(dtRNAs) are hairpin-shaped RNA structures placed on the 5' untranslated region of the mRNA, and they could modulate the degradation rate constant of prokaryotic mRNA by resisting endocellular RNase attack. This part is one of the coding sequences of dtRNA published by Zhang et al.in 2021. dtRNA1 is the first-ranking dtRNA in the fluorescence measurements, indicating this dtRNA has a relatively strong ability to resist mRNA degradation from endocellular RNases in E.coli.

This part type could increase the yield of expressed products without posting an extra metabolic burden to the host cell since it facilitates product accumulation by decreasing degradation rather than enhancing gene expression. For protein products such as GFP reporters, it regulates the dynamic range of concentration up to several folds. For functional RNA products, the effect is much more prominent since the anti-degradation effect on mRNA is more direct. In principle, this type of part could be used in distinct research directions in synthetic biology. For example, dtRNA could improve the yield of valuable products in biosynthesis by circumventing the trade-off between gene expression and excessive cellular pressure. With the help of dtRNAs, it is possible for advanced genetic circuits with enhanced complexity to work in living systems, eventually promoting the materialization of arbitrarily-designed artificial organisms.

Scheme: Functions and downstream applications of dtRNAs

dtRNAs are compact in size(10-60 nucleotides). For usage, they are compatible with most assembly methods that use overlapping primers containing dtRNA coding sequences and accessorial adaptor sequences as integration fragments in HiFi assembly, Golden Gate assembly, and Biobrick assembly.

Characterization by 2022 team HUS_United

This year our team attempted to introduce the newly published degradation-tuning RNAs as a powerful toolbox to the iGEM community. To test the probability of iteratively designing dtRNAs, we designed a second-generation dtRNA(dtRNA1v2) with optimal structural parameters based on the structure of dtRNA1 and used NUPACK to simulate the secondary structure.

Figure 1: structure of dtRNA1 predicted by NUPACK

1. Stem length: 11 bp; Structural factor: 11/11=100%

2. Stem GC content: 54.5%; Structural factor: 54.5/60=90%

3. Loop size: 6 nt; Structural factor: 6/6=100%

Then we experimentally integrate dtRNA1v2 into GFP-expressing cassettes under the control of medium strength promoter J23106 and medium RBS B0032, then measure the fluorescence and OD value, then compare it with the original version of dtRNA1. The samples for measurement are prepared as follows:

1. Use inverse PCR to obtain the vector fragment, and the position of the joint is right upstream of the RBS B0032.

2. Commercially purchased single-stranded DNA integration fragments(promoter sequences) with two homologous arms are inserted into the linearized vector through HiFi assembly. (Approximately 50:1 in molar ratio)

3. E.coli DH5α competent cells are transformed with finished HiFi assembly reactions, and colonies are picked and sequenced on the next day.

4. Colonies with correct sequences are cultured for 8h and then transferred to a microplate with 100-fold dilution, GFP fluorescence (excitation=488 nm, emission=515 nm) and OD600 are measured every 15 minutes for 16 h duration.

The results are as follows:

Figure 2: Fluorescence curve of dtRNA1v2

The result shows a slightly higher fluorescence value compare to the original version of dtRNA1, with the GFP fold change up to 3.96. Although this value may not be statistically significant when adding repeating groups, it shows the potential of designing second-generation dtRNAs using forward-engineering principles. All in all, we think we have demonstrated our engineering success by using in silico simulation to guide our design and experiments and applying rational design principles to further optimize our basic parts.

Sequence and Features