Team:MIT/L7AeRepressingSystem

• BACKGROUND
• EXPERIMENTS
  Promoters miRNA Recombinase Combining parts Collaborations
• FUTURE WORK & DESIGN
  Circuit Design Clinical Application
• HUMAN PRACTICES
  Accessibility Integrated Human Practices Public Outreach
• TEAM
  Team Acknowledgements
• NOTEBOOK
  Lab Notebook Basic Parts Parts collection Attributions Safety

L7Ae - Kink turn

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RNA-Based Gene Regulation

L7Ae, an archaeal ribosomal protein, binds with high affinity to RNA motifs called kink-turns (K-turns), found in both archaeal and eukaryote RNAs [1][2][3]. L7Ae protein sequence is divided into three structural regions consisting of a highly conserved RNA-binding region (RBR) flanked by less conserved N-terminal and C-terminal regions [2]. Variation in the terminal regions could dictate RNA-binding specificity of different homologs of L7Ae protein [2]. When a K-turn motif is inserted into the target mRNA upstream of the open reading frame, L7Ae can be used as a translational regulator [1][2][3]. The binding activity of L7Ae will prevent the ribosome machinery from performing translation. The strength of the repression can be controlled by varying the distance between the K-turns and the 5’-end of the mRNA, or by changing the number of the k-turn motifs [1].

Figure. Binding of L7Ae to kink-turn motifs preveting translation.

Recombinase and L7Ae-Kturn

Purpose

Using recombinases as biological latches giving our genetic circuit the ability to memorize disease temporal specificity. However, since the recombinase is controlled by an inducible promoter, leaky expression of the promoter (activation without input signals - disease biomarkers) could lead to unwanted activation of the output gene. By puting k-turn motifs in front of the recombinase gene, we hope to reduce leaky expression of the recombinase when the system is inactivated.
We designed an experiment to examine the repression level of the L7Ae - kink turn system on the expression of an output gene (EYFP - Enhanced yellow flourescent protein), which is regulated by TP901 (a serine recombinase).

Experimental Setup

Read more about building kturn constructs here.

Result

Testing the effect of varying k-turn sequences

REFERENCE:

1. Oliwia Andries, Tasuku Kitada, Katie Bodner, Niek N Sanders & Ron Weiss (2015) Synthetic biology devices and circuits for RNA-based ‘smart vaccines’: a propositional review, Expert Review of Vaccines, 14:2, 313-331
2. Gagnon KT, Zhang X, Qu G, et al. Signature amino acids enable the archaeal L7Ae box C/D RNP core protein to recognize and bind the K-loop RNA motif. Rna 2010;16(1):79-90
3. Stapleton JA, Endo K, Fujita Y, et al. Feedback control of protein expression in mammalian cells by tunable synthetic translational inhibition. ACS Synth Biol 2012;1(3):83-8