2.3 Implementation with transcriptional circuits
2.3.2 Cross-activation
2.3.2.3 Materials and methods
The DNA strands were designed using the Winfree lab DNA design toolbox for MATLAB, Nupack [2] and Mfold [141], following the constraint guidelines in [61], Chapter 3.4. All the strands were purchased from Integrated DNA Technologies, Coralville, IA [1]. T1−nt is labeled with TYE 563 at the 50 end, T2−nt is labeled with TYE665 at the 50 end, both activators A1 and A2 are labeled with the IOWA black RQ quencher at the 30 end. T7 RNAP was purchased from Epicentre Biotechnologies, Cat. n. TM910K (200 U/µl). E. coli cloned RNase H was purchased from Ambion, Cat. n. AM2292 (10 U/µl). Inorganic lyophilized pyrophosphatase added to the transcription
Act Inh
Enz
A
B
C
0 200 400
0 50 100
[nM]
Time (min) Active T1
0 200 400
0 50 100
[nM]
Time (min) Active T2
Act RNAP
RNase H Act RNAP
RNase H
0 50 100 150
0 20 40 60 80 100
Time (min)
[nM]
[T1](0)=50 nM [T2](0)=100 nM
0 50 100 150
0 50 100 150
Time (min)
[nM]
[T1](0)=50 nM [T2](0)=150 nM
0 50 100 150
0 50 100 150 200
Time (min)
[nM]
[T1](0)=50 nM [T2](0)=200 nM
0 50 100 150
0 20 40 60 80 100
Time (min)
[nM]
[T1](0)=100 nM [T2](0)=50 nM
0 50 100 150
0 50 100 150
Time (min)
[nM]
[T1](0)=150 nM [T2](0)=50 nM
0 50 100 150
0 50 100 150 200
Time (min)
[nM]
[T1](0)=200 nM [T2](0)=50 nM
0 50 100 150
0 20 40 60 80 100
Time (min)
[nM]
[T1](0)=100 nM [T2](0)=100 nM
0 1 2 3 4 5
0 1 2 3 4 5
Ratio of total amount of genelets
Final ratio of active genelets
T2 varied, T
1 fixed T1 varied, T
2 fixed No regulation Numerical model
Figure 2.17: A. The current design for the transcriptional cross-activation scheme suffers from a self-inhibition side reaction. The two circuits were here considered in isolation. Self-inhibition is particularly strong for the genelet T2, right panel. Note that adding RNase H to the active genelet T2 does not allow recovery of the fully-on state; we do not have a good explanation for this behavior yet. B. Fluorescent traces showing the behavior of the two genelets simultaneously in solution, for different total amounts of templates. [Atoti ] = 300 nM, [Itoti ] = 1µM in all the experimental traces. Dashed lines represent numerically computed traces, using model (2.13) and the adjusted parameters in Table 2.4. C. Plot showing the steady-state ratio versus total ratio of the genelets for the data collected at B.
protocol was purchased from Sigma Aldrich, Cat. n. I1891-100UN.
Transcription protocol
The transcription buffer mix was prepared prior to each experiment run (for four samples), mixing reagents to the following final concentrations: 1x Transcription Buffer and 10 mM dithiothreitol (DTT) (Epicentre Biotechnologies, Cat. n. BP1001), 7.5 mM each rNTP (Epicentre Biotechnologies, Cat. n. RN02825), 35 mM MgCl2, and 0.015 U/µl pyrophosphatase (resuspended in Tris HCl 20 mM, pH 7.2, 50% glycerol (v/v)). The templates were annealed in 1×Epicentre transcription buffer from 90◦C to 37◦C for 1 h 30 min at a concentration 5–10×the target concentration. The annealed templates, DNA activators, and inhibitors were added to the transcription buffer mix and incubated at 37◦C. Each transcription experiment for fluorescence spectroscopy was prepared for a total target volume of 60µl. Enzymes were pre-mixed before each experimental run (two or four samples), with a volume ratio of 5:1, and 4µL of the pre-mix was added to each cuvette.
Data acquisition
All fluorescence experiments were performed on a Horiba/Jobin Yvon Fluorolog 3 system, using 45 µL sample chamber quartz cuvettes. Fluorescence was measured at 37◦ C every two minutes.
Excitation/emission for TYE563 (T1) were set to the maxima 549–563 nm, those for TYE665 (T2) at 645–665 nm, as recommended by the manufacturer IDT DNA. Slit widths were set to 2 nM for excitation and 4 nM for emission. The raw measured data were converted to estimated switch activity by normalizing with respect to maximum fluorescence Φmax (measured before adding activators, inhibitors and enzymes) and to minimum fluorescence Φmin (measured after adding activators and before adding inhibitors and enzymes):
[TiAi](t) = [Ttoti ]·
1−v(t)·Φ(t)−Φmin Φmax−Φmin
,
wherev(t) is a correction factor that takes into account the percent volume dilution introduced by the addition of activators and inhibitors. For the data in Figure 2.17 A, v = 1. For the data in Figure 2.17 B and C, after the addition of activators and inhibitors, v(t) is taken to be 1.15; after addition of enzymesv(t)=1.2. (Dilution was very high due to the low concentration of stock aliquots and to the high target concentration of activators and inhibitors.)
Numerical simulations
The system was numerically analyzed using MATLAB (The MathWorks). Differential equations were solved using the ode23 routine. The initial numerical studies to obtain some insight on the circuit behavior were performed using the parameters in Table 2.3, which are consistent with [63]
and the references cited therein. After data collection, a subset of the parameters was tuned. The adjusted parameters are the rates kTiAi, kAiIi, kTiAiIi,kRiAiIi, kRiAi,kRiRj, kRiTj, and the parameters kcatONii, kcatOFFi, kcatOFFij; their values are in Table 2.4. The total amount of RNAP was fixed at
80 nM, the total amount of RNase H at 11 nM (to reflect on the higher volume of RNase H used in this set of experiments, relative to the negative auto-regulation project).
Oligonucleotides sequences
Due to technical constraints of the supplier IDT DNA, T1-nt and T2-nt were shortened (with respect to the nominal design) to a length of 100 bases. These modifications do not alter the regulatory domains of the transcripts. The full length of the main transcription products was not affected, as verified by gel electrophoresis (data not shown). A2 and I2, V1 were used for the experiment in Figure 2.17 A. A2 and I2, V2 were instead used for the all the experiments in Figure 2.17 B and C.
T1-nt Full (121-mer) 5’-CAT TAG TGT CGT TCG TTC ATA ATA CGA CTC ACT ATA GGG AGA AGT GGT TAA GGT ATA GTT AGA TAG GTA AGG CAT GTT CAT TAG TGT CGT TGT GTA GTG TTG CTG ACT AAA AGT CAG AAA A-3’ (not synthesized)
T1-nt Short (99-mer) 5’-TYE563-CAT TAG TGT CGT TCG TTC ATA ATA CGA CTC ACT ATA GGG AGA AGT GGT TAA GGT ATA GTT AGA TAG GTA AGG CAT GTT CAT TAG TGT CGT TGT GTA GTG -3’
T1-t (97-mer) 5’-TTT TCT GAC TTT TAG TCA GCA ACA CTA CAC AAC GAC ACT AAT GAA CAT GCC TTA CCT ATC TAA CTA TAC CTT AAC CAC TTC TCC CTA TAG TGA GTC G-3’
T2-nt Full (116-mer) 5’- CCT TAC CTA TCT AAC TAT ATA ATA CGA CTC ACT ATA GGG AGA CAA CAC TAC ACT GAA CGA ACG ACA CTA ATG AAC ATG CCT TAC CTA TCT ACC TTA ACC ACT TGA CAA AGT CAA AA-3’ (not synthesized)
T2-nt Short (99-mer) 5’- TYE665-CCT TAC CTA TCT AAC TAT ATA ATA CGA CTC ACT ATA GGG AGA CAA CAC TAC ACT GAA CGA ACG ACA CTA ATG AAC ATG CCT TAC CTA TCT ACC TTA ACC -3’
T2-t(92-mer) 5’-TTT TGA CTT TGT CAA GTG GTT AAG GTA GAT AGG TAA GGC ATG TTC ATT AGT GTC GTT CGT TCA GTG TAG TGT TGT CTC CCT ATA GTG AGT CG-3’
A1 (30-mer) 5’-TAT TAT GAA CGA ACG ACA CTA ATG AAC TAC-IOWA black RQ-3’
I1(36-mer) 5’-GTA GTT CAT TAG TGT CGT TCG TTC AGT GTA GTG TTG-3’
A2 V1 (30-mer) 5’-TAT TAT ATA GTT AGA TAG GTA AGG CAT TAG-IOWA black RQ-3’
I2V1 (36-mer) 5’-CTA ATG CCT TAC CTA TCT AAC TAT ACC TTA ACC ACT-3’
A2V2 (33-mer) 5’-TAT TAT ATA GTT AGA TAG GTA AGG CAT TAG TAG -IOWA black RQ-3’
I2V2 (39-mer) 5’-CTA CTA ATG CCT TAC CTA TCT AAC TAT ACC TTA ACC ACT -3’