Difference between revisions of "Part:BBa K1368010"

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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1368010 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K1713020 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K1368010 parameters</partinfo>
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<partinfo>BBa_K1713020 parameters</partinfo>
 
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== Usage and Biology ==
 
== Usage and Biology ==
 +
 +
<p>Imaging RNA in living cells is important for understanding the function and regulation of divece classes of cellular RNAS. There are some aptamers like Spinach and dBroccoli,witch bind and activates the fluorescence of fluorophores similar to that found in green fluorescent protein. In our project, At first, we use GFP as a reporter of our oscillator, but there is a long delay from DNA to protein. We think it’s better to shorten the delay. Luckily, there is a RNA aptamer named dBroccoli that can be used for the genetic encoding of fluorescent RNA. The dBroccoli binds and activates the fluorescence of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1Himidazol-5(4H)-one) (DFHBI-1T).</p>
  
 
<p>DMHBI was synthesized as outlined inScheme.</p>
 
<p>DMHBI was synthesized as outlined inScheme.</p>
−
[[File:HZAU2014-荧光素1.png|800px]]
+
[[File:HZAU2015-荧光素1.png|800px]] （荧光素反应1）
 +
（荧光素反应2）
 +
 
 +
<p>N-Acetylglycine
 +
(2.22 g, 18.99 mmol), anhydrous sodium acetate (1.56 g, 18.99 mmol), 4-hydroxy-3,5-difluorobenzaldehyde (3.00 g, 18.99 mmol), and acetic anhydride (7.2 ml) were stirred at 100 ºC for 1 h. After allowing the reaction to cool to room temperature, cold ethanol (30ml) was added while stirring and the reaction was left stirring overnight at 4 ºC. The resulting crystalline solid was then washed with a small amount of cold ethanol, hot water, hexanes and dried to afford 3.40 g (yield 64%) of 1 as a pale yellow solid。Compound 1 (472 mg, 1.68 mmol) was refluxed with 4 ml of ethanol, 250 mg (2.52
 +
mmol) of 2,2,2-Trifluoroethylamine, and 348 mg of potassium carbonate for 4 h. The
 +
reaction mixture was removed from heat and cooled to room temperature. The solvent
 +
was evaporated and the mixture was redissolved in a 1:1 mixture of ethyl acetate and 500
 +
mM sodium acetate pH 3.0. The organic layer was separated, dried with anhydrous
 +
sodium sulfate. The solvent was removed under reduced pressure and the reaction
 +
mixture was purified by column chromatography (CH2Cl2 : MeOH = 10:1) to afford 215
 +
mg (yield 40%) of 12 as yellow solid。</p>
  
−
<p>N-Acetylglycine (5.26 g, 0.045mol), anhydrous sodium acetate (3.69 g,0.045mol), 4-hydroxy-2,3-dimethoxybenzaldehyde (8.19 g, 0.045mol), and aceticanhydride (15 ml) were stirred at 90℃for 2 h. After allowing the reaction to coolto room temperature, cold ethanol (20 ml) was added while still stirring andthe reaction was left stirring overnight at 4℃.The resultingcrystalline solid was then washed with coldethanol, hot water, hexanesand driedto afford9.65 g (yield 70%) of 3as a pale yellow solid:H NMR (500 MHz,) δ7.56 (s, 2H), 7.18 (s,1H), 3.98 (s, 6H), 2.54(s, 3H), 2.39(s, 3H); LC/MS (LC: gradient 20-95% MeCN[0.1%] over 2.5min, 0.5 ml/minflow rate, MS:): retention time, 2.15 min;purity, 95%; 306.36. Then wecan get Compound 3.Compound 3 (1.12 g, 0.005mol) was refluxedwith 15 ml of ethanol, 1 ml of 40% aqueous methylamine, and 700 mg of potassiumcarbonate for 4h. The reaction mixturewas removed from heat and upon cooling formed an orange precipitate. Theprecipitate containing the product was filtered and washed briefly with coldethanol. The precipitate was then redissolved in a 1:1 mixture of ethyl acetateand 500 mM sodium acetate pH 3.0. The organic layer was separated, dried withanhydrous sodium sulfate and solvent was removed under reduced pressure toyield 717 mg (yield 52%) of DMHBI as an orange solid: 1H NMR (500MHz, DMSO-) δ7.62 (s,2H), 6.90 (s, 1H), 3.80 (s, 6H), 3.09 (s, 3H), 2.34 (s, 3H).DMHBI is easily oxidized. So storagerequires low temperature, dark and inert gas environment.</p>
+
<p>DH5α cells (TransGen Biotech) were transformed with 10 μl of plasmid DNA expressing dBrpccoli. Cells were plated, grown overnight and single colonies were picked for inoculation overnight in Luria Broth containing chloramphenicol. At OD600 = 0.8, 200 μl culture was removed, pelleted and resuspended in 200 μl M9 minimal media with 200 μM DFHBI-1T and cultivate for 40 min at 37 ℃. we observed the spinach RNA aptamer by the fluorescence microscope, the result is in the figure 1.</p>
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<p>DH5α cells (TransGen Biotech) were transformed with 10 μl of plasmid DNA expressing chimeras of the human tRNALys3 scaffold fused to the Spinach aptamer sequence. Cells were plated, grown overnight and single colonies were picked for inoculation overnight in Luria Broth containing chloramphenicol. At OD600 = 0.8, 150 μl culture was removed, pelleted and resuspended in 100 μl M9 minimal media and cultivate for 1 h at 37 ℃. Cells were washed twice and incubated with 200 μM DFHBI in M9 media for 5 min. We tested the spinach RNA aptamer in the Tanon 1600R Gel Imaging System under the 302nm, the control groups are the DMHBI and DMHBI + control RNA. The result is below (Fig.1) and we also observed the spinach RNA aptamer by the fluorescence microscope, the result is in the figure 2.</p>
+
  
  
−
[[File:HZAU2014-RNA 适配子1.png|430px|thumb|left|Fig. 1 photographed by Gel Imaging System under the 302nm]]
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[[File:HZAU2014-RNA 适配子1.png|430px|thumb|left|Fig. 1photographed by the fluorescence microscope (40X)]]
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[[File:HZAU2014-RNA 适配子_2.png|445px|thumb|right|Fig. 2 photographed by the fluorescence microscope (40X)]]
+
  
  
 
<p >References</p>
 
<p >References</p>
−
<p>Paige J S, Wu K Y, Jaffrey S R. RNA mimics of green fluorescent protein[J]. Science, 2011, 333(6042): 642-646.</p>
+
<p>Wenjiao S, Strack R L, Nina S, et al. Plug-and-play fluorophores extend the spectral properties of Spinach.[J]. Journal of the American Chemical Society, 2014, 136(4):1198-1201.
 +
Filonov G S, Moon J D, Nina S, et al. Broccoli: Rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution.[J]. Journal of the American Chemical Society, 2014, 136.</p>

Revision as of 20:24, 18 September 2015

Spinach Aptamer 13-2 RNA driven by c1 promoter

Spinach Aptamer 13-2 RNA is a small RNA with specific stem loop structure which can combine with DMHBI, a kind of fluorescein, and then the RNA-fluorophore complexes will fluoresce under the excitation of ultraviolet light. We use cI promoter to derive Spinach Aptamer 13-2 RNA that the part will serve as a reporter in transcription level under control of cI protein which acts as an input signal.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Usage and Biology

Imaging RNA in living cells is important for understanding the function and regulation of divece classes of cellular RNAS. There are some aptamers like Spinach and dBroccoli,witch bind and activates the fluorescence of fluorophores similar to that found in green fluorescent protein. In our project, At first, we use GFP as a reporter of our oscillator, but there is a long delay from DNA to protein. We think it’s better to shorten the delay. Luckily, there is a RNA aptamer named dBroccoli that can be used for the genetic encoding of fluorescent RNA. The dBroccoli binds and activates the fluorescence of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1Himidazol-5(4H)-one) (DFHBI-1T).

DMHBI was synthesized as outlined inScheme.

800px （荧光素反应1） （荧光素反应2）

N-Acetylglycine (2.22 g, 18.99 mmol), anhydrous sodium acetate (1.56 g, 18.99 mmol), 4-hydroxy-3,5-difluorobenzaldehyde (3.00 g, 18.99 mmol), and acetic anhydride (7.2 ml) were stirred at 100 ºC for 1 h. After allowing the reaction to cool to room temperature, cold ethanol (30ml) was added while stirring and the reaction was left stirring overnight at 4 ºC. The resulting crystalline solid was then washed with a small amount of cold ethanol, hot water, hexanes and dried to afford 3.40 g (yield 64%) of 1 as a pale yellow solid。Compound 1 (472 mg, 1.68 mmol) was refluxed with 4 ml of ethanol, 250 mg (2.52 mmol) of 2,2,2-Trifluoroethylamine, and 348 mg of potassium carbonate for 4 h. The reaction mixture was removed from heat and cooled to room temperature. The solvent was evaporated and the mixture was redissolved in a 1:1 mixture of ethyl acetate and 500 mM sodium acetate pH 3.0. The organic layer was separated, dried with anhydrous sodium sulfate. The solvent was removed under reduced pressure and the reaction mixture was purified by column chromatography (CH2Cl2 : MeOH = 10:1) to afford 215 mg (yield 40%) of 12 as yellow solid。

DH5α cells (TransGen Biotech) were transformed with 10 μl of plasmid DNA expressing dBrpccoli. Cells were plated, grown overnight and single colonies were picked for inoculation overnight in Luria Broth containing chloramphenicol. At OD600 = 0.8, 200 μl culture was removed, pelleted and resuspended in 200 μl M9 minimal media with 200 μM DFHBI-1T and cultivate for 40 min at 37 ℃. we observed the spinach RNA aptamer by the fluorescence microscope, the result is in the figure 1.


Fig. 1photographed by the fluorescence microscope (40X)


References

Wenjiao S, Strack R L, Nina S, et al. Plug-and-play fluorophores extend the spectral properties of Spinach.[J]. Journal of the American Chemical Society, 2014, 136(4):1198-1201. Filonov G S, Moon J D, Nina S, et al. Broccoli: Rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution.[J]. Journal of the American Chemical Society, 2014, 136.