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. 1 Image of Pseudovibrio denitrificans colonies in a petri dish containing kanamycin. Pseudovibrio denitrificans were previously transformed with the pNK2-CRPIIh plasmid.
   Pd corresponds to Pseudovibrio denitrificans no transformed
   Pd1, Pd2, Pd3, Pd4, Pd5 correspond to Pseudovibrio denitrificans transformed
   
   
   
   
   
   Fig. 2 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. 3 Picture of an electrophorese gel samples come from the PCR which amplify the kanamycineR cassette. All clones have the insertion of the transposon. The Control, Pseudovibrio denitrificans, do not the insertion as expected.
   
   
   b) Integration transposon in Pseudovibrio denitrificans
   
   Here, we tried to proved that there was actually a random integration of the transposon in the strain's genome. To achieve this, we performed a reverse PCR.
   How many bands appear ? A question which relates to the number of inserts in the genome. What is the size and numbers of bands between genomic DNA samples? A question which relates to potential discovery of favourable spots of integration in the genome and the randomness of the number of insertions in the genome.
   
   
   Fig. 4 Schema of reverse PCR
   
   In order to verify both number and frequency of insertion per transformation we set up the following protocol:
   Thanks to the genome assembly we were able to digest the genomic DNA in silico and find a restriction enzyme that would not cut in our insert but cut enough times in the genome to be able to recircularize pieces of DNA.
   HindIII cuts the genome generates small fragment ready for religation.
   After the recircularization we looked for and enzyme that could cut in our insert but with the least number of knacks in the genome sequence in order to cut only once the circular DNAs.
   We used XbaI because it exhibits the lowest number of cut in the gene and does not cut our insert.
   
   With the relinearize fragment we can perform a PCR to find:
   
   
   Fig. 5 Picture of electrophorese gel from reverse PCR in Pseudovibrio denitrificans which integrated the transposon.
   
   One clone clearly displayed numerous insertions, while the others having fewer bands tend to show band sof the same size, which could prefigure the existence of favourite spot, as can be seen in E.coli.
   With further experiments and sequencing run we will be able to precisely locate the insertions and the better define how many times an insertion event can occur at a given concentration of plasmid electroporated.
   
   
   The transformation of Pseudovibrio denitrificans has been verified by the study of its phenotype and its genotype. We therefore confirm the efficiency of the transposon system in our bacteria.
   
   
   
   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.
   
   
   
   Fig. 6 Picture of electrophoresis gel of Transposon plasmid isolated to pSB1C3

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