Part:BBa_K3027000
A1 is a nuclease coming from T5 phage. We have demonstated that this biobrick can eliminate genomic DNA and thus be used to efficiently generate DNA-less chassis.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Nuclease_A1 sequence (556 amino acids):
MVISAEKQTVILKMAADFNFYGKRLRATKLEVCDDISKMVYDTTKHSTAICDWLEANKPAKPKAAKVAKAIKNDERPEAAGIVSSTVEQWEVKQGKRFIITSIQNNTFPHKNFLASLEQYAQFIGADLLVSKFIYNKNGFQNGEGADGIRYDSAFDKYICNKNVFLNNRRFAFMAEINVLPTADYPLSGFAETATALNLEGLAIGAAKITAESVPALKGEIVRRMYSTGTATLKNYIQQKAGQKAEALHNFGALIVEFDEDGEFFVRQLETMDESGVFYDLNTCATPAGCYETTGHVLGLQYGDIHAEKLDEECAAASWGHGDTYGLVDILKPKYQFVHDVHDFTSRNHHNRASGVFLAKQYAAGRDKVLDDLIDTGRVLESMERDFSQTIIVESNHDLALSRWLDDRNANIKDDPANAELYHRLNAAIYGAIAEKDDTFNVLDYALRKVAGCEFNAIFLTTDQSFKIAGIECGVHGHNGINGSRGNPKQFKKLGKLNTGHTHTASIYGGVYTAGVTGSLDMGYNVGASSWTQTHIITYANGQRTLIDFKNGKFFA
To characterize the nuclease, its CDS has been inserted in a plasmid under the control of an inducible promoter. This plasmid was then used to transform bacteria which will express the nuclease for the study.
Plasmid Description
The pBAD24 cloning vector contains an ampicillin resistance gene, the pBR322_origin for replication, an araC gene encoding the AraC transcriptional regulator and the arabinose-inducible PBAD promoter (also called Para). We modified plasmid pBAD24 to introduce two BsaI sites downstream the PBAD promoter to facilitate cloning using the Golden Gate technique. This new plasmid, named pBAD24-MoClo, allows type IIs cloning of a coding sequence (CDS) with the prefix AATG and the suffix GCTT.
The nuclease A1 gene (BBa_K3027000) was inserted in pBAD24-Moclo using the two BsaI sites to obtain the plasmid pBAD24-nuclease_A1.
In the absence of arabinose, AraC forms a homodimer that represses transcription of the gene beneath the control of PBAD. Arabinose modifies the conformation of AraC homodimer to activate PBAD-dependent transcription. AraC synthesis is also controlled by the presence of glucose in the media. At low glucose concentration, the adenylate cyclase produces cAMP, which associates with CRP to activate araC transcription. Therefore, maximum expression from the promoter PBAD is reached when bacteria are grown in the absence of glucose and the presence of arabinose.
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