Biomod/2014/UCR/Breaking RNA/Acknowledgements: Difference between revisions

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<u>Unbound Aptamer-Kleptamer Interaction</u>
<u>Unbound Aptamer-Kleptamer Interaction</u>
<br>
<br>
An important factor in the oscillatory system is to ensure the R1 and K1 strands are not strongly interacting before the inhibition of SP6 RNAP. In this gel, the R1 and K1 aptamers were mixed into solution together at the same concentration and incubated. For both variations of the K1 strand (23&38 bp), there are two distinct bands in lanes 4 and 6. This indicates that there are little interactions occurring between these two strands before R1 binds to the enzyme. This can be explained by a difference in secondary structure between the bound and unbound forms of R1.
An important factor in the oscillatory system is to ensure the R1 and D1 strands are not strongly interacting before the inhibition of SP6 RNAP. In this gel, the R1 and D1 aptamers were mixed into solution together at the same concentration and incubated. For both variations of the D1 strand (23&38 bp), there are two distinct bands in lanes 4 and 6. This indicates that there are little interactions occurring between these two strands before R1 binds to the enzyme. This can be explained by a difference in secondary structure between the bound and unbound forms of R1.


[[Image:20140807 nativegel sp6 38nt 23nt sp6&38 sp6&23 T7new&RNAP T7oldRNAPedited.png|350px|thumb|center|<font size="1.5">Non-denaturing gel electrophoresis of aptamer and kleptamer interactions. Lanes 2-4 represent the aptamer, D1 (38 base pair), and D1 (23 base pair), respectively. Lanes 5 and 6 correspond to the aptamer along with D1 (23 base pair) and D1 38 base pair, respectively.</font>]]
[[Image:20140807 nativegel sp6 38nt 23nt sp6&38 sp6&23 T7new&RNAP T7oldRNAPedited.png|350px|thumb|center|<font size="1.5">Non-denaturing gel electrophoresis of aptamer and kleptamer interactions. Lanes 2-4 represent the aptamer, D1 (38 base pair), and D1 (23 base pair), respectively. Lanes 5 and 6 correspond to the aptamer along with D1 (23 base pair) and D1 38 base pair, respectively.</font>]]

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  <li><a href='http://openwetware.org/wiki/Biomod/2014/UCR/Breaking_RNA'><span>Home</span></a></li>
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Supplement

EDIT


Oligonucleotide Sequences

1.1 Oscillator Genes and Strands

DNA Oligonucleotides Length Sequences
G1 Non-Template 83 5'-TAA TAC GAC TCA CTA TAG GAT GGC AGC GGA GAG TTG CTT GGA ATG CGT TAT AGT CTC TTA GGT GTG TTC GCA CAC CAC TCT CC-3'
G1 Template 83 5'-GGA GAG TGG TGT GCG AAC ACA CCT AAG AGA CTA TAA CGC ATT CCA AGC AAC TCT CCG CTG CCA TCC TAT AGT GAG TCG TAT TA-3'
G2 Non-Template 56 5'-ATT TAG GTG ACA CTA TAG AGG CGA GAA GCA GCA ATG ATA GTG GAA TTG ACT TAC GC-3'
G2 Template 56 5'-GCG TAA GTC AAT TCC ACT ATC ATT GCT GCT TCT CGC CTC TAT AGT GTC ACC TAA AT-3'
G3 Non-Template 52 5'-TTC TAA TAC GAC TCA CTA TAG CGT AAG TCA ATT CCA CTA TCA TTG CTG CTT C-3'
G3 Template 52 5'-GAA GCA GCA ATG ATA GTG GAA TTG ACT TAC GCT ATA GTG AGT CGT ATT AGA A-3'
G4 Non-Template 83 5'-ATT TAG GTG ACA CTA TAG AGG CGA CGT CGC CTC TCA ACG AAC CTT ACG CAA TAT CAG AGA ATC CAC ACA AGC GTG TGG TGA GAG G-3'
G4 Template 83 5'-CCT CTC ACC ACA CGC TTG TGT GGA TTC TCT GAT ATT GCG TAA GGT TCG TTG AGA GGC GAC GTC GCC TCT ATA GTG TCA CCT AAA T-3'
D1 61 5'-TexRd-CGT CGC CTC TCA ACG AAC CTT ACG CAA TAT CAG AGA ATC CAC ACA AGC GTG TGG TGA GAG G-IowaBlack-3'
​

1.2 Bistable Genes and Strands

DNA Oligonucleotides Length Sequences
G1 Non-Template 83 5'-TAA TAC GAC TCA CTA TAG GAT GGC AGC GGA GAG TTG CTT GGA ATG CGT TAT AGT CTC TTA GGT GTG TTC GCA CAC CAC TCT CC-3'
G1 Template 83 5'-GGA GAG TGG TGT GCG AAC ACA CCT AAG AGA CTA TAA CGC ATT CCA AGC AAC TCT CCG CTG CCA TCC TAT AGT GAG TCG TAT TA-3'
G2(B) Non-Template 57 5'-ATT TAG GTG ACA CTA TAG AGG CGA GCG TAA GTC AAT TCC ACT ATC ATT GCT GCA AGC-3'
G2(B) Template 57 5'-GCT TGC AGC AAT GAT AGT GGA ATT GAC TTA CGC TCG CCT CTA TAG TGT CAC CTA AAT-3'
D1 100 5'-TexRd-CGT CGC CTC TCA ACG AAC CTT ACG CAA TAT CAG AGA ATC CAC ACA AGC GTG TGG TGA GAG G-IowaBlack-3'
Spinach Non-Template 100 5'-ATT TAG GTG ACA CTA TAG AGG ACG CGA CCG AAA TGG TGA AGG ACG GGT CCA GTG CTT CGG CAC TGT TGA GTA GAG TGT GAG CTC CGT AAC TGG TCG CGT C-3'
Spinach Template 100 5'-GAC GCG ACC AGT TAC GGA GCT CAC ACT CTA CTC AAC AGT GCC GAA GCA CTG GAC CCG TCC TTC ACC ATT TCG GTC GCG TCC TCT ATA GTG TCA CCT AAA T-3'
Malachite Green Non-Template 68 5'-ACT ATG ATA ATA CGA CTC ACT ATA GGG AGA GGA TCC CGA CTG GCG AGA GCC AGG TAA CGA ATG GAT CC-3'
Malachite Green Template 68 5'-GGA TCC ATT CGT TAC CTG GCT CTC GCC AGT CGG GAT CCT CTC CCT ATA GTG AGT CGT ATT ATC ATA GT-3'
​


Characterization of Spinach and Malachite Green aptamer

EDIT

Aptamer-Dye Titrations

Figure 2.1: SP aptamer (A) and MG aptamer (B) titration after aptamer concentration was increased from 2 μM to 3 μM. More than 10 hours after SP aptamer addition was needed before stable fluorescence was reached, while only approximately 2 hours was needed after the MG aptamer addition.

Aptamer-dye titration experiments were performed to correlate fluorescent intensity levels to dye-aptamer levels. Two types of titrations were performed: one with aptamers as the titrant and another with fluorescent dye as the titrant. . Aside from the original premise, these experiments uncovered some challenges in using MG and Spinach as reporters.

For the aptamer titrations, New England Biolabs RNA Polymerase Transcription Buffer, Nuclease Free Water, and excess Spinach and MG dye (20 μM each) were combined in a 0.5 mL tube and transferred to a cuvette for measurement in a spectrofluorometer. Varying amounts of extracted Spinach and MG aptamer were added to the cuvette and stirred for approximately 20 seconds. Fluorescence was measured until intensity remained relatively constant. This data shows that both the Spinach and MG aptamers do not bind to their respective dyes quickly after being added. SP took significantly longer than MG to reach a stable fluorescent intensity after the concentration of both aptamers in the solution was increased from 2 μM to μM (Figure 2.1). These results suggest Spinach is unsuitable as a reporter due to the large time lag. However, MG still may be a viable, albeit not as effective as molecular beacons, as described in the results.

Figure 2.2: Mapping and comparison of SP Dye titration done on 9/2/14 and 9/9/14. Data indicates that SP dye fluorescence levels are not consistent at high concentrations of dye.

Dye titrations were also performed with SP dye. Prior to the titration, a 60 μL transcription was prepared by combining nuclease-free water, 7.5 μM NTP, 24 mM MgCl2, 200 nM spinach gene, 1x NEB RNA Polymerase Transcription Buffer, and 4 μL SP6 RNAP. Solution was incubated at 30 degrees Celsius for 24 hours to produce an abundance of SP aptamer. Ten μL of this transcript solution was then diluted to 60 uL and transferred into a cuvette and placed in a spectrofluorometer. Varying known concentrations of Spinach dye was added to the solution. After each Spinach dye addition, fluorescent intensity was measured for 15 minutes after fluorescent intensity stabilized. Intensities for each dye addition were averaged. Intensity and dye concentration were plotted and mapped (figure 2.2). The results of the dye titration experiments indicate that intensities are consistent at low dye concentrations up to 3 μM, but vary greatly with higher concentrations. This is further evidence that Spinach may not be a good reporter to use for our systems. .

Fluorometer Experiments On Circuit Components

Reactivation of T7 RNA Polymerase with Genelets
EDIT In this case, the genes- G3 and G2 –were used instead of the aptamers. There is obvious inhibition of the enzyme with the addition of varying concentrations of G3. After a few hours, G2 (750 nM) was added to the mixture to be transcribed into the reactivator, R2. Unfortunately, there was no reactivation of the enzyme. It’s possible this may be because the transcription of G3 is outcompeting the transcription of G2. This would prevent any reactivation since T7 RNAP would immediately re-inhibit itself.

Reactivation attempt of 7 RNA Polymerase using g2.

Gel Experiments On Circuit Components

Unbound Aptamer-Kleptamer Interaction
An important factor in the oscillatory system is to ensure the R1 and D1 strands are not strongly interacting before the inhibition of SP6 RNAP. In this gel, the R1 and D1 aptamers were mixed into solution together at the same concentration and incubated. For both variations of the D1 strand (23&38 bp), there are two distinct bands in lanes 4 and 6. This indicates that there are little interactions occurring between these two strands before R1 binds to the enzyme. This can be explained by a difference in secondary structure between the bound and unbound forms of R1.

Non-denaturing gel electrophoresis of aptamer and kleptamer interactions. Lanes 2-4 represent the aptamer, D1 (38 base pair), and D1 (23 base pair), respectively. Lanes 5 and 6 correspond to the aptamer along with D1 (23 base pair) and D1 38 base pair, respectively.

Modeling

Switch

EDIT

TEXT
TEXT.

Table 1 displays the estimated conditions that were used to simulate the bistable switch. Realistic parameters were assumed for kinetics and concentrations to simulate actual experimental conditions. This suggests that it is possible to construct a functional in vitro bistable switch.

Clock

EDIT

TEXT
TEXT.
TEXT.
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