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| {{UCR/Breaking-RNA/HEAD}}
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| <font size="5">
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| <h1> Supplement </h1>
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| </font>
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| [http://openwetware.org/index.php?title=Biomod/2014/UCR/Breaking_RNA/Acknowledgements&action=edit EDIT]
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| <div id='submenu'>
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| <ul>
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| <li>[[#Oligonucleotide Sequences | Oligonucleotide Sequences]]</li>
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| <li>[[#Characterization of Spinach and Malachite Green aptamer| Characterization of Spinach and Malachite Green aptamer]]</li>
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| <li>[[#Reactivation of T7 RNA Polymerase with Genelets| Reactivation of T7 RNA Polymerase with Genelets]]</li>
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| <li>[[#Modeling | Modeling]]</li>
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| </ul>
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| </div>
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| <br />
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| <font size="4.5">
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| <h2>Oligonucleotide Sequences</h2>
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| </font>
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| <h3><u><font size="4.5">1.1 Oscillator Genes and Strands</font></u></h3>
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| ::{| border = "2" class="wikitable" style="margin: 1em auto 1em auto;"
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| |-
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| ! width = "20%" | DNA Oligonucleotides
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| ! width = "10%" | Base Pair Length
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| ! width = "70%" | Sequences
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| |- align="center"
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| | '''G1''' Non-Template
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| | 83
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| | align="left" | 5'-<span style="color:red">TAA TAC GAC TCA CTA TAG GAT G</span>GC AGC GGA GAG TTG CTT GGA ATG CGT TAT AGT CTC TTA GGT GTG TTC GCA CAC CAC TCT CC-3'
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| |- align="center"
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| | '''G1''' Template
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| | 83
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| | align="left" | 5'-GGA GAG TGG TGT GCG AAC ACA CCT AAG AGA CTA TAA CGC ATT CCA AGC AAC TCT CCG CTG C<span style="color:red">CA TCC TAT AGT GAG TCG TAT TA</span>-3'
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| |- align="center"
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| | '''G2''' Non-Template
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| | 56
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| | align="left" | 5'-<span style="color:red">ATT TAG GTG ACA CTA TAG AGG CGA</span> GAA GCA GCA ATG ATA GTG GAA TTG ACT TAC GC-3'
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| |- align="center"
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| | '''G2''' Template
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| | 56
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| | align="left" | 5'-GCG TAA GTC AAT TCC ACT ATC ATT GCT GCT TC<span style="color:red">T CGC CTC TAT AGT GTC ACC TAA AT</span>-3'
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| |- align="center"
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| | '''G3''' Non-Template
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| | 52
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| | align="left" | 5'-<span style="color:red">TTC TAA TAC GAC TCA CTA TA</span>G CGT AAG TCA ATT CCA CTA TCA TTG CTG CTT C-3'
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| |- align="center"
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| | '''G3''' Template
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| | 52
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| | align="left" | 5'-GAA GCA GCA ATG ATA GTG GAA TTG ACT TAC GC<span style="color:red">T ATA GTG AGT CGT ATT AGA A</span>-3'
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| |- align="center"
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| | '''G4''' Non-Template
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| | 83
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| | align="left" | 5'-<span style="color:red">ATT TAG GTG ACA CTA TAG AGG CGA</span> CGT CGC CTC TCA ACG AAC CTT ACG CAA TAT CAG AGA ATC CAC ACA AGC GTG TGG TGA GAG G-3'
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| |- align="center"
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| | '''G4''' Template
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| | 83
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| | align="left" | 5'-CCT CTC ACC ACA CGC TTG TGT GGA TTC TCT GAT ATT GCG TAA GGT TCG TTG AGA GGC GAC G<span style="color:red">TC GCC TCT ATA GTG TCA CCT AAA T</span>-3'
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| |- align="center"
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| | '''k1'''
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| | 61
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| | align="left" | 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'
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| |}
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| <h3><u><font size="4.5">1.2 Bistable Genes and Strands</font></u></h3>
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| ::{| border = "2" class="wikitable" style="margin: 1em auto 1em auto;"
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| |-
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| ! width = "20%" | DNA Oligonucleotides
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| ! width = "10%" | Base Pair Length
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| ! width = "70%" | Sequences
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| |- align="center"
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| | '''G1''' Non-Template
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| | 83
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| | align="left" | 5'-<span style="color:red">TAA TAC GAC TCA CTA TAG GAT G</span>GC AGC GGA GAG TTG CTT GGA ATG CGT TAT AGT CTC TTA GGT GTG TTC GCA CAC CAC TCT CC-3'
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| |- align="center"
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| | '''G1''' Template
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| | 83
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| | align="left" | 5'-GGA GAG TGG TGT GCG AAC ACA CCT AAG AGA CTA TAA CGC ATT CCA AGC AAC TCT CCG CTG C<span style="color:red">CA TCC TAT AGT GAG TCG TAT TA</span>-3'
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| |- align="center"
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| | '''G2(B)''' Non-Template
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| | 57
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| | align="left" | 5'-<span style="color:red">ATT TAG GTG ACA CTA TAG AGG CGA</span> GCG TAA GTC AAT TCC ACT ATC ATT GCT GCA AGC-3'
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| |- align="center"
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| | '''G2(B)''' Template
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| | 57
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| | align="left" | 5'-GCT TGC AGC AAT GAT AGT GGA ATT GAC TTA CGC <span style="color:red">TCG CCT CTA TAG TGT CAC CTA AAT</span>-3'
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| |- align="center"
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| | '''k1'''
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| | 100
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| | align="left" | 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'
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| |- align="center"
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| | '''Spinach''' Non-Template
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| | 100
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| | align="left" | 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'
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| |- align="center"
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| | '''Spinach''' Template
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| | 100
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| | align="left" | 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'
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| |- align="center"
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| | '''Malachite Green''' Non-Template
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| | 68
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| | align="left" | 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'
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| |- align="center"
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| | '''Malachite Green''' Template
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| | 68
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| | align="left" | 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'
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| |}
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| <font size="4.5">
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| <h2> Characterization of Spinach and Malachite Green aptamer </h2>
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| </font>
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| [http://openwetware.org/index.php?title=Biomod/2014/UCR/Breaking_RNA/Acknowledgements&action=edit EDIT]
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| <p><u><font size="4.5">Binding of MG and SP Aptamers</font></u></p>
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| [[Image:SP_one_titration.png|500px|thumb|center|<font size="1.5">SP aptamer titration after aptamer concentration was increased from 2 uM to 3uM. More than 10 hours after aptamer addition was needed before stable fluorescence was reached.]]
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| [[Image:MG_one_titration.png|500px|thumb|center|<font size="1.5">MG aptamer titration after aptamer concentration was increased from 2 uM to 3uM. Approximately 2 hours was needed for fluorescence to stabilize.]]
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| <p>Titrations of SP and MG aptamers were done to correlate fluorescent intensity levels to aptamer concentration. However, these experiments also show the rates that the aptamers bind to the dye. New England Biolabs RNA Polymerase Transcription Buffer, Nuclease Free Water, and 20 &u;M (excess) SP and MG dye were combined in a 0.5 mL tube and transferred to a cuvette for measurement in a spectrofluorometer. Varying amounts of SP 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 SP 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 uM to 3uM (Figure). These results suggest SP 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.</p>
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| <u>Unbound Aptamer-Kleptamer Interaction</u>
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| <br>
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| 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.
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| [[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.]]
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| <font size="4.5">
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| <u>Reactivation of T7 RNA Polymerase with Genelets </u>
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| </font>
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| <br>
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| [http://openwetware.org/index.php?title=Biomod/2014/UCR/Breaking_RNA/Acknowledgements&action=edit EDIT]
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| 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.
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| [[Image:Data 20140923.png|350px|thumb|center|<font size="1.5">Reactivation attempt of 7 RNA Polymerase using g2.]]
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| <u>Comparison of Transcription Rates between SP6 RNAP and T7 RNAP </u>
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| [[Image:20141017_Transcription Speeds of SP6 and T7.jpg|350px|thumb|center|<font size="1.5">Denaturing gel electrophoresis of SP6 RNAP transcribing gene g2 and T7 RNAP transcribing gene g1. Lane 5-8 represent aptamer r2, gene g2, aptamer r1, gene g1, respectively. Lane 1-3 represent SP6 RNAP transcribing gene g2 at 100nm, 150nm, and 200nm, respectively. Lane 4 represent T7 RNAP transcribing gene g1 at 10nm.]]
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| <font size="4.5">
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| <h2> Modeling </h2>
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| </font>
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| <font size="4.5">
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| <h3> Switch </h3>
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| </font>
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| [http://openwetware.org/index.php?title=Biomod/2014/UCR/Breaking_RNA/Acknowledgements&action=edit EDIT]
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| [[Image: ToggleEquations.jpg|upright=8|thumb|center|<font size="1.5">TEXT]]
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| [[Image: ToggleEquilibriumConditions.jpg|upright=8|thumb|center|<font size="1.5">TEXT.]]
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| [[Image: ToggleTable.jpg|upright=4|thumb|center|<font size="1.5">TEXT.]]
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| <font size="4.5">
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| <h3> Clock </h3>
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| </font>
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| [http://openwetware.org/index.php?title=Biomod/2014/UCR/Breaking_RNA/Acknowledgements&action=edit EDIT]
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| [[Image: ClockEquations.jpg|upright=8|thumb|center|<font size="1.5">TEXT]]
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| [[Image: ClockEquilibriumConditions.jpg|upright=8|thumb|center|<font size="1.5">TEXT.]]
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| [[Image: ClockTable.jpg|upright=4|thumb|center|<font size="1.5">TEXT.]]
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