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L7Ae - Kink turn

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

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

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.