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Revision as of 16:10, 19 October 2016

L7Ae k-turn repressing system

L7Ae - Kink turn

Back to recombinase overview page

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

Purpose

Experimental Setup

                                     

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

Untransfected ControlSingle 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
                   
Untransfected ControlSingle 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