Team:MIT/L7AeRepressingSystem

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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]. The 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

We talked with other graduate students in Dr. Weiss' lab to gain more information about the L7Ae/k-turn repressing system, and learned that high concentration of L7Ae could potentially be harmful for mammalian cells since mammalian cell cultures have been showing unhealthy morphology under high concentration of L7Ae. Thus, we designed an experiment to examine the effect of L7Ae on HEK293.

Experimental Setup

L7Ae was put under an inducible pTRE/Dox promoter system. Higher amount of Dox (a small molecul inducer ) would increase the amount of L7Ae in the cell. The reporter gene for L7Ae was pTRE:mKate. Yellow flourescent (hEF1a:EYFP) was the transfection marker. The cells were induced by different amount of Dox concentration 24hrs post transfection. The cells were trypsinzed and harvested for Flow Cytomertry analysis 24hrs after induction. We also use SYTOX blue for live-dead cells analysis.

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: Dead cells.

At low cell count, the amount of dead cells is constant when Dox is less than 1000nM. When Dox is higher than 1000nM, the amount of dead cells increases linearly, indicating the cells becoming unhealthy. At high cell count, the amount of cell dead increases in a logarithmic fashion and saturates at Dox = 1000nM. Thus, we decided to use 0-1000nM for the Dox concentration range to regulate the expression of L7Ae.

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.

We used 2 inducible promoter systems - pEGSH/PonA and pTRE/Dox - in this experiment to control the expression of two genes, L7Ae for tuning the repressing level and TP901, thre recombinase. The pTRE/Dox system controled the expression of L7Ae. We induced the cells with Dox at the same time as transfection because the repressing system needed to be activated before TP901 recombinase was induced by PonA/pEGSH.
Additionally, including all the neccessary genes (TP901, flipped EYFP, L7Ae, VgEcr-RXR, and rtTA) and the reporter flourescent genes, the total number of plasmids went up to 7 (or 8 with the nude DNA - pDONR). We were reaching the maximum limitation of plasmid number that could be transfected using lipofection. After asking for advice from other graduate students in the lab, we increased the ratio of viafect:DNA from 1ul:500ng total DNA to 1.5ul:500ng total DNA.
For each well:
Total amount of DNA: 1500ng
Viafect transfection reagent: 4.5ul

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.

Read more about pEGSH/PonA inducible promoter system here.

Result

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

Figure. Dox activates pTRE controlling the expressiong of L7Ae. PonA = 0uM: uninduced TP901 recombinase. PonA = 5uM: induced TP901 recombinase.

These graphs show that as the amount of Dox increased, there was smaller amount of EYFP being activated when TP901 was induced. When Dox = 0uM, at high transfection efficiency, the amount of activated EYFP in induced TP901 sample was 2-fold higher than in uninduced TP901 sample (y-axis is in log scale). However, when Dox = 1000nM, the amount of activated EYFP in uninduced and induced TP901 samples stayed the same for a larger range of transfection efficiency. However, at high transfection efficiency, the ammount of activated EYFP is still higher for induced TP901 than uninduced; and the ammount difference is larger at lower concentration of Dox. Thus, L7Ae/k-turn RNA-based gene regulation system could reduce the basal expresion of inactivated TP901, but still allow the recombinase to perform its function when the whole system activated.

Testing the effect of varying k-turn sequences

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

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

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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
4. Wroblewska et al. Mammalian synthetic circuits with RNA binding proteins for RNA-only delivery. Nature Biotechnology 33, 839–841 (2015)