Difference between revisions of "Team:MIT/L7AeRepressingSystem"
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Revision as of 17:28, 19 October 2016
L7Ae - Kink turn
Back to recombinase overview pageRNA-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
| 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.
| 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:
- 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
- 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
- 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
