RNA

Part:BBa_K4511006

Designed by: Wenhe Ma   Group: iGEM22_HUS_United   (2022-10-12)
Revision as of 14:34, 12 October 2022 by Bioche (Talk | contribs) (→‎Characterization by 2022 team HUS_United)


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.


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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