Difference between revisions of "Part:BBa K3380153"
 
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The Broccoli aptamer was designed and described by [https://pubs.acs.org/doi/abs/10.1021/ja508478x Filonov ''et al.'' (2014)].
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The Broccoli aptamer was designed and described by [https://pubs.acs.org/doi/abs/10.1021/ja508478x Filonov ''et al.'' (2014)]. ''''Figure 1''' shows the Broccoli secondary structure folding prediction as simulated by [http://www.nupack.org// NUPACK].
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The Broccoli secondary structure folding prediction as simulated by [http://www.nupack.org// NUPACK] is shown below.  
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{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
 
{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
 
! colspan="2" style="background:#C0C0C0"|'''Broccoli Characteristics'''
 
! colspan="2" style="background:#C0C0C0"|'''Broccoli Characteristics'''
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|DFHBI-1T
 
|DFHBI-1T
 
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|-
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|'''Excitation wavelength'''
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|'''Excitation wavelength (nm) '''
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|472 (nm)
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|472  
 
|-
 
|-
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|'''Emission wavelength '''
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|'''Emission wavelength (nm) '''
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|507 (nm)
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|507  
 
|-
 
|-
 
|'''Quantum Yield'''
 
|'''Quantum Yield'''
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|-
 
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|'''Extinction coefficient'''
 
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|29600 (M^-1 cm-1)
 
 
|0.94
 
|0.94
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|-
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|'''Extinction coefficient (M^-1 cm-1)'''
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|29600
 
|-
 
|-
 
|'''Source'''
 
|'''Source'''
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[[File:Part BBa K3380153.png|400px|Broccoli folding]]
 
[[File:Part BBa K3380153.png|400px|Broccoli folding]]
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'''Figure 1: Broccoli secondary structure prediction.''' The adenine (A), cytosine (C), guanine (G) and uracil (U) nucleobases are shown in green, blue, black and red respectively.
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===Usage and Biology===
 
===Usage and Biology===
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It is usually used in live-cell imaging, however it can also be used in vitro. The Broccoli aptamer can be used for visualisation by gene fusion to the target of interest. Its fluorescent colour is green. The most commonly used fluorophore in tandem with Broccoli aptamer is DFHBI-1T ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4H)-one). The DFHBI-1T fluorophore has an excitation wavelength of 472 (nm) and an emission wavelength of 507 (nm).
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It is usually used in live-cell imaging, however it can also be used in vitro. The Broccoli aptamer can be used for visualisation by gene fusion to the target of interest. Its fluorescent colour is green. The most commonly used fluorophore in tandem with Broccoli aptamer is DFHBI-1T ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4H)-one).  
  
 
Its short length allows it to be synthesized rather than being cloned using plasmids. To increase its stability and increase its folding the Broccoli can be transcribed using tRNA scaffolds such as F30 (BBa_K3380101 and BBa_K3380102). The tRNA scaffolds protect the fluorescent aptamers from the degradation by RNAses, therefore the scaffolds increases their fluorescence. However, Broccoli can be successfully used without a scaffold.  
 
Its short length allows it to be synthesized rather than being cloned using plasmids. To increase its stability and increase its folding the Broccoli can be transcribed using tRNA scaffolds such as F30 (BBa_K3380101 and BBa_K3380102). The tRNA scaffolds protect the fluorescent aptamers from the degradation by RNAses, therefore the scaffolds increases their fluorescence. However, Broccoli can be successfully used without a scaffold.  
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<span class='h3bb'>'''Sequence and Features'''</span>
 
<span class='h3bb'>'''Sequence and Features'''</span>
 
<partinfo>BBa_K3380153 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3380153 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3380153 parameters</partinfo>
 
<partinfo>BBa_K3380153 parameters</partinfo>
 
<!-- -->
 
<!-- -->
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==Contribution ==
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*'''Group:''' [https://2021.igem.org/Team:SCU-China SCU-China iGEM team 2021]
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*'''Author:''' Yilong Xu
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*'''Summary:''' Improve this part by expanding itself
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*'''Link to our biobrick: '' https://parts.igem.org/Part:BBa_K3977006
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===Characterization/Improvement ===
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Based on BBa_K3380153, we construct a RNA adaptor with 4 repeats of BBa_K3380153,
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We put them under J23100, and detect their performance in 488/527.
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Our result shows that BBa_K397706 show more output (C) , meaning that it performed better than original part BBa_K3380153 and our improvement is successful.
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[[image: T--SCU-China--scu-2021-4X.png|600px|center|]]
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                        Figure 1: Relative fluorescence intensity BBa_K3380153 and BBa_K3977006

Latest revision as of 21:39, 21 October 2021


Broccoli fluorescent RNA aptamer

Broccoli is a fluorescent RNA aptamer comprising a short ribonucleotide sequence that exhibits fluorescence upon binding to a specific fluorophore.

Characterization

The Broccoli aptamer was designed and described by Filonov et al. (2014). 'Figure 1 shows the Broccoli secondary structure folding prediction as simulated by [http://www.nupack.org// NUPACK].

Broccoli Characteristics
Fluorophore DFHBI-1T
Excitation wavelength (nm) 472
Emission wavelength (nm) 507
Quantum Yield 0.94
Extinction coefficient (M^-1 cm-1) 29600
Source [1]


Broccoli folding

Figure 1: Broccoli secondary structure prediction. The adenine (A), cytosine (C), guanine (G) and uracil (U) nucleobases are shown in green, blue, black and red respectively.


Usage and Biology

It is usually used in live-cell imaging, however it can also be used in vitro. The Broccoli aptamer can be used for visualisation by gene fusion to the target of interest. Its fluorescent colour is green. The most commonly used fluorophore in tandem with Broccoli aptamer is DFHBI-1T ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4H)-one).

Its short length allows it to be synthesized rather than being cloned using plasmids. To increase its stability and increase its folding the Broccoli can be transcribed using tRNA scaffolds such as F30 (BBa_K3380101 and BBa_K3380102). The tRNA scaffolds protect the fluorescent aptamers from the degradation by RNAses, therefore the scaffolds increases their fluorescence. However, Broccoli can be successfully used without a scaffold.

To increase its fluorescence, the Broccoli dimerization has been successfully attempted, exhibiting almost twice the fluorescence of the monomeric aptamer.

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]



Contribution

Characterization/Improvement

Based on BBa_K3380153, we construct a RNA adaptor with 4 repeats of BBa_K3380153, We put them under J23100, and detect their performance in 488/527.

Our result shows that BBa_K397706 show more output (C) , meaning that it performed better than original part BBa_K3380153 and our improvement is successful.

T--SCU-China--scu-2021-4X.png
                        Figure 1: Relative fluorescence intensity BBa_K3380153 and BBa_K3977006