Part:BBa_K4511000

dtRNA1

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. Although several reports are showing that special secondary structures at the 5’ UTR region of mRNA would resist degradation from endogenous RNase, these structures are not comprehensively designed and tested until the report published by Zhang et al. in 2021. We thought these degradation-tuning structures might be perfect materials for modulating degradation. We used NUPACK to predict the secondary structure of dtRNAs, and compare the key structural parameters with correspondent optimal conditions.

Figure 1: structure of dtRNA1 predicted by NUPACK

1. Stem length: 12 bp; Structural factor: 11/12=91.7%

2. Stem GC content: 66.7%; Structural factor: 60/66.7=90%

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

Relative GFP expression in E.coli DH10B: 4.801

For wet lab work, we integrate six dtRNA structures into expression cassettes under the control of the J23106 promoter with medium strength.

Figure 1: Schematic diagram of gene circuit

After HiFi assembly, transformation, and microplate reading, the obtained fluorescent curves and growth curves are used to characterize the anti-degradation effect of dtRNAs and the metabolic pressure, respectively. We found that changes in dtRNA structure led to negligible deviation in the cell growth curve but a significant range in fluorescence. Compared with the previous report from the literature, we observed comparable fluorescent signals, indicating the effect of degradation-tuning RNAs is indeed transferrable to DH5α strains, the only difference is that the introduction of dtRNA82 did not abolish the GFP fluorescence, which may come from the difference of Rnase targeting sites between the two strains.

Figure 2: GFP fluorescent curve under the control of J23106

Figure 3: Comparison of dtRNA fluorescent fold change with different host strains

On the other hand, the measured OD600 curve shows a similar but even faster growth curve when using dtRNAs, no matter whether its function is to resist or facilitate degradation. This interesting observation deserves further investigation in the future. These experiment results solidified dtRNAs’ ability to change expressions and confirm our simulation results of not posting burden to the host.

Figure 4: Bacterial growth curve under the control of J23106

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]