Difference between revisions of "Part:BBa K2457002"

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<partinfo>BBa_K2457002 short</partinfo>
 
<partinfo>BBa_K2457002 short</partinfo>
 
<p>BBa_K2457002 was engineered to be a building block of the standard BioBrick toolbox for bacterial genome: the ([http://2017.igem.org/Team:Amazonas_Brazil pCRISPeasy]). It’s the first BioBrick available on Registry with an improved sgRNA sequence - both crRNA design and tracrRNA secondary structure - to increase the CRISPR-Cas9 editing efficiency.  BBa_K2457002 is composed by: the J23100 constitutive promoter sequence + an optimized sgRNA (crRNA with a GG motif at the 3’ end of it’s target-specific sequence from LacZ [1] and tracrRNA with extended duplex length and a mutation at the thymine 4 [2]) + L3S2P21 terminator [3]. It was rationally designed to be standardized AND interchangeable, aiming to provide a BioBrick to attend multiple genome editing purposes. For that matter, It’s easy to engineer new modules aiming to insert information into the genome through RFC10 Assembly.</p>
 
<p>BBa_K2457002 was engineered to be a building block of the standard BioBrick toolbox for bacterial genome: the ([http://2017.igem.org/Team:Amazonas_Brazil pCRISPeasy]). It’s the first BioBrick available on Registry with an improved sgRNA sequence - both crRNA design and tracrRNA secondary structure - to increase the CRISPR-Cas9 editing efficiency.  BBa_K2457002 is composed by: the J23100 constitutive promoter sequence + an optimized sgRNA (crRNA with a GG motif at the 3’ end of it’s target-specific sequence from LacZ [1] and tracrRNA with extended duplex length and a mutation at the thymine 4 [2]) + L3S2P21 terminator [3]. It was rationally designed to be standardized AND interchangeable, aiming to provide a BioBrick to attend multiple genome editing purposes. For that matter, It’s easy to engineer new modules aiming to insert information into the genome through RFC10 Assembly.</p>
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[[File:BBa K2457002 circuit.png]]
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Figure 1: BBa_K2457002 circuit.
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===Usage and Biology===
 
===Usage and Biology===

Revision as of 17:00, 1 November 2017


Optimized sgRNA targeting LacZ

BBa_K2457002 was engineered to be a building block of the standard BioBrick toolbox for bacterial genome: the ([http://2017.igem.org/Team:Amazonas_Brazil pCRISPeasy]). It’s the first BioBrick available on Registry with an improved sgRNA sequence - both crRNA design and tracrRNA secondary structure - to increase the CRISPR-Cas9 editing efficiency. BBa_K2457002 is composed by: the J23100 constitutive promoter sequence + an optimized sgRNA (crRNA with a GG motif at the 3’ end of it’s target-specific sequence from LacZ [1] and tracrRNA with extended duplex length and a mutation at the thymine 4 [2]) + L3S2P21 terminator [3]. It was rationally designed to be standardized AND interchangeable, aiming to provide a BioBrick to attend multiple genome editing purposes. For that matter, It’s easy to engineer new modules aiming to insert information into the genome through RFC10 Assembly.

BBa K2457002 circuit.png

Figure 1: BBa_K2457002 circuit.


Usage and Biology

Single-guide RNA (sgRNA) is one of the two building blocks of the CRISPR-Cas9 genome editing machinery, which directs the Cas9 protein to introduce double-stranded breaks (DSB) into target genomes sequences.

Design

Characterization

BBa K2457002 electropherogram.png Figure 2: Sequencing electropherogram from BBa_K2457002.

BBa K2457002 alignment.png

Figure 3: Alignment of the designed sequence and our final construction from BBa_K2457002.


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]