Part:BBa_K1413044:Experience

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Applications of BBa_K1413044

Application of pNK2 which is the original plasmid :

Insertion of transposon

1. Transformation Pseudovibrio denitrificans with pNK2
   
   We tested pNK2 by transforming Pseudovibrio denitrificans bacteria with the plasmid by electroporation. The selection of cells was performed in 1X marine broth medium supplemented with kanamycine 50µg/mL. In fact, Pseudovibrio denitrificans can grow on medium with kanamycine 25µg/mL. (Sensitivity to antibiotics)
   
   
   
2. Phenotypic verification
   
   
   
   Fig. 3 Image of Pseudovibrio denitrificans colonies in a petri dish containing kanamycin. Pseudovibrio denitrificans were previously transformed with the pNK2-CRPIIh plasmid.
   
   
   
   
   
   Fig. 4 Growth curve of Pseudovibrio denitrificans in M9 supplemented with casamino acids and 3% NaCl
   
   
3. Genotypic verification
   
   
   a) Amplification of transposon
   
   The kanamycin gene is included in the transposon sequence of pNK2 and we thus amplify this gene for our verification PCR.
   We successfully obtained an amplicon corresponding to the kanamycin gene in our transformed cells. Considering the plasmids are not able to be replicated in an other strain than pir cells, we assumed the amplification we obtained were from ADNg and not from plasmids.
   Fig. 5 Picture of an electrophorese gel samples come from the PCR which amplify the kanamycineR cassette. All clones have the insertion of teh transposon. The Control, Pseudovibrio denitrificans, do not the insertion as expected.
   
   As a first verification, we obtained this gel for 15 colonies of Pseudovibrio denitrificans.
   
   
   
   align="center">b) Sequencing of 16S
   
   The amplification of transformed cells' DNA with primers 16S, which amplify the sequence of the ribosome 16S, were purified and sent to sequencing.
   The sequence we obtained proves that we transformed actual Pseudovibrio bacteria with an integrated transposon.
   
   
   
   
   c) Amplification of specific sequence of Pseudovibrio denitrificans
   After the results of sequencing of Pseudovibrio denitrificans' genome, a sequence specific of this strain has been discovered. Application of BBa_K1413044 : To replicate this new plasmid we can used E. coli.
   To separate the pSB1C3 and new pNK2 we must used BglII. After that the disgest product must migrate in the electrophoresis gel.The band to 2,6 kb , which is the biggest correspond to transposon plasmid. The transposon plasmid can be extract in the gel. The T4 ligase is used to circularize the plasmid.
   All the biobrick can introduce between the Is10 with biobrick enzyme. Also this biobrick can be integrate in Pseudovibrio denitrificans the genome with electro transformation. The voltage used is 2000V and the incubation time after the electroporation is to 3h.
   
   
   Picture of electrophoresis gel of Transposon plasmid isolated to pSB1C3

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