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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.

Toxicity concentration of L7Ae in mammalian cell

Experimental Setup

Click here to show/hide plate map of L7Ae toxicity experiment.                                      

Untransfected Control	Single color (Y) 500ng hEF1a:eYFP 500ng pDONR	Single color (R) 500ng hEF1a:mKate 500ng pDONR	Single color (B) 500ng hEF1a:tagBFP 500ng pDONR
Three colors 300ng hEF1a:eYFP 300ng hEF1a:mKate 300ng hEF1a:BFP 100ng pDONR	300ng TRE:L7Ae 300ng TRE:mKate 100ng hEF1a:rTta 100ng hEF1a:eYFP 200ng pDONR 0nM Dox	300ng TRE:L7Ae 300ng TRE:mKate 100ng hEF1a:rTta 100ng hEF1a:eYFP 200ng pDONR 20 nM Dox	300ng TRE:L7Ae 300ng TRE:mKate 100ng hEF1a:rTta 100ng hEF1a:eYFP 200ng pDONR 50 nM Dox
300ng TRE:L7Ae 300ng TRE:mKate 100ng hEF1a:rTta 100ng hEF1a:eYFP 200ng pDONR 100 nM Dox	300ng TRE:L7Ae 300ng TRE:mKate 100ng hEF1a:rTta 100ng hEF1a:eYFP 200ng pDONR 200 nM Dox	300ng TRE:L7Ae 300ng TRE:mKate 100ng hEF1a:rTta 100ng hEF1a:eYFP 200ng pDONR 500 nM Dox	300ng TRE:L7Ae 300ng TRE:mKate 100ng hEF1a:rTta 100ng hEF1a:eYFP 200ng pDONR 1000 nM Dox
300ng TRE:L7Ae 300ng TRE:mKate 100ng hEF1a:rTta 100ng hEF1a:eYFP 200ng pDONR 2000 nM Dox

Result

Figure. Testing the toxicity of L7Ae in HEK293. Varying amount of Dox would vary the expression of L7Ae (Dox concentraions: 1, 20, 50, 100, 200, 500, 1000, 2000 nM). X-axis: hEF1a:EYFP = transfection marker, Y-axis: TRE:mKate = L7Ae induced by pTRE.

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

Figure. Diagram explaining experimental setup testing the effect of L7Ae/k-turn repressing system on basal expression of recombinase TP901.

Click here to show/hide plate map of TP901 - L7Ae/k-turn experiment.                    

Untransfected Control	Single color (Y) 1000ng hEF1a:eYFP 500ng pDONR	Single color (R) 1000ng hEF1a:mKate 500ng pDONR	Single color (B) 1000ng hEF1a:tagBFP 500ng pDONR
Three colors 500ng hEF1a:eYFP 500ng hEF1a:mKate 500ng hEF1a:BFP	Control no L7Ae 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 0ng TRE: L7Ae 200ng hEF1a:BFP 300ng pDONR 1000nM Dox; 5uM PonA	Control no k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 1000nM Dox; 0uM PonA	Control no k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 1000nM Dox; 5uM PonA
Experiment 2x k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 0nM Dox; 0uM PonA	Experiment 2x k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 0nM Dox; 5uM PonA	Experiment 4x k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 0nM Dox; 0uM PonA	Experiment 4x k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 0nM Dox; 5uM PonA
Experiment 2x k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 100nM Dox; 0uM PonA	Experiment 2x k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 100nM Dox; 5uM PonA	Experiment 4x k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 100nM Dox; 0uM PonA	Experiment 4x k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 100nM Dox; 5uM PonA
Experiment 2x k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 500nM Dox; 0uM PonA	Experiment 2x k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 500nM Dox; 5uM PonA	Experiment 4x k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 500nM Dox; 0uM PonA	Experiment 4x k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 500nM Dox; 5uM PonA
Experiment 2x k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 1000nM Dox; 0uM PonA	Experiment 2x k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 1000nM Dox; 5uM PonA	Experiment 4x k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 1000nM Dox; 0uM PonA	Experiment 4x k-turn 300ng EGSH-kturn: TP901 300ng EGSH-kturn:mKate 200ng hEF1a: flipped EYFP 100ng hEF1a: VgEcr 100ng hEF1a: rtTA 100ng TRE: L7Ae 200ng hEF1a:BFP 1000nM Dox; 5uM PonA

Read more about building kturn constructs here.

Result

Testing the 2x k-turn L7Ae system with varied L7Ae expression level

Figure.

Testing the effect of varying k-turn sequences

Figure. Dox = 1000nM - activate the L7Ae/k-turn repressing system, and PonA = 0uM - activate the expression of TP901 recombinase.

Figure. Dox = 1000nM - activate the L7Ae/k-turn repressing system, and PonA = 5uM - activate the expression of TP901 recombinase.

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