Part:BBa_K2457003
Standardized RecA coding sequence
The BioBrick BBa_K2457002 is an essential building block part to constructions that will be involved in 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 direction, being composed by: the RecA coding sequence and the L3S2P00 transcription terminator.
Figure 1: BBa K2457003 circuit.
Usage and Biology
After the cleavage by Cas9 in the target sequence at a ribonucleoprotein complex with the guide RNA, blunt-end strands are originated. In SOS response, the endogenous RecBCD repair enzymes from the cell machinery degrade the blunt-end extremities in 5’ to 3’ direction, up to 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 search for 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:
Design
Characterization
BBa_K2457000 was used by Amazonas_Brazil team in CROUT experiments and CRIN experiments.
CROUT (CRISPeasy-out method): Designed for knock-out strategy for bacterial genome editing based on CRISPR/Cas9
RESULTADOS DE CROUT AQUI JU
CRIN (CRISPeasy-in method): Designed for knock-in strategy for bacterial genome editing based on CRISPR/Cas9
RESULTADOS DE CRIN AQUI JU
Sequencing
Figure 2:Sequencing electropherogram from BBa_K2457003.
Figure 3:Alignment of the designed sequence and our final construction from BBa_K2457003.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 508
Illegal AgeI site found at 1012 - 1000COMPATIBLE WITH RFC[1000]
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