Difference between revisions of "Part:BBa K2457003"

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<p>After the cleavage by Cas9 in the target sequence at a ribonucleoprotein complex with the guide RNA, originating blunt-end strands. In SOS response, the endogenous RecBCD repair enzymes from the cell machinery degrade the blunt-end extremities in 5’ to 3’ direction until the chi site recognition. Then, their activity in 5’ to 3’ direction is finished and keeps going to 3’ strand, forming 3’ DNA single stick-ends strands. This segment is recognized by RecA, which builds a DNA-protein filament able to investigate for a homologous template on undamaged DNA sequences, assisting on the polymerase invasion process and setting-up the Holliday Junction. This generates a crossover with the strands, leading to the recombination among the undamaged strand and the broken strand.  
 
<p>After the cleavage by Cas9 in the target sequence at a ribonucleoprotein complex with the guide RNA, originating blunt-end strands. In SOS response, the endogenous RecBCD repair enzymes from the cell machinery degrade the blunt-end extremities in 5’ to 3’ direction until the chi site recognition. Then, their activity in 5’ to 3’ direction is finished and keeps going to 3’ strand, forming 3’ DNA single stick-ends strands. This segment is recognized by RecA, which builds a DNA-protein filament able to investigate for a homologous template on undamaged DNA sequences, assisting on the polymerase invasion process and setting-up the Holliday Junction. This generates a crossover with the strands, leading to the recombination among the undamaged strand and the broken strand.  
 
Now let's analyze the functional scheme of this building block:</p>
 
Now let's analyze the functional scheme of this building block:</p>
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===Disign===
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[[File:BBa K2457003 circuit.png]]
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Figure 1: BBa K2457003 circuit.
  
 
===Characterization===
 
===Characterization===
  
 
[[File:BBa K2457003 electropherogram.png]]
 
[[File:BBa K2457003 electropherogram.png]]
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Figure 1:Sequencing electropherogram from BBa_K2457003.  
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Figure 2:Sequencing electropherogram from BBa_K2457003.  
  
 
[[File:BBa K2457003 aligmnent.png]]
 
[[File:BBa K2457003 aligmnent.png]]
  
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Figure 2:Alignment of the designed sequence and our final construction from BBa_K2457003.
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Figure 3:Alignment of the designed sequence and our final construction from BBa_K2457003.
  
 
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Revision as of 09:42, 1 November 2017


Standardized RecA coding sequence

Description


Usage and Biology

The BioBrick BBa_K2457002 is an essential building block part to constructions that will be involved with homologous recombination processes. It was developed by Amazonas_Brazil team in 2017 to be a CRISPeasy vector building block: a standard BioBrick vector to bacterial genome engineering. This part has the length of 1210bp and it is oriented in reverse, being composed of the RecA coding sequence and the L3S2P00 transcription terminator.


After the cleavage by Cas9 in the target sequence at a ribonucleoprotein complex with the guide RNA, originating blunt-end strands. In SOS response, the endogenous RecBCD repair enzymes from the cell machinery degrade the blunt-end extremities in 5’ to 3’ direction until the chi site recognition. Then, their activity in 5’ to 3’ direction is finished and keeps going to 3’ strand, forming 3’ DNA single stick-ends strands. This segment is recognized by RecA, which builds a DNA-protein filament able to investigate for a homologous template on undamaged DNA sequences, assisting on the polymerase invasion process and setting-up the Holliday Junction. This generates a crossover with the strands, leading to the recombination among the undamaged strand and the broken strand. Now let's analyze the functional scheme of this building block:

Disign

BBa K2457003 circuit.png

Figure 1: BBa K2457003 circuit.

Characterization

BBa K2457003 electropherogram.png Figure 2:Sequencing electropherogram from BBa_K2457003.

BBa K2457003 aligmnent.png

Figure 3:Alignment of the designed sequence and our final construction from BBa_K2457003.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 508
    Illegal AgeI site found at 1012
  • 1000
    COMPATIBLE WITH RFC[1000]