Part:BBa_K1413044

A fusion of Transposon Plasmid and pSB1C3

This part is composed of OriVR6Kgamma (BBa_K1314041), Tn10 (BBa_K1314043) and 2 sites Is10 (repeat inverse sequences recognized by the transposase Tn10). This part can be used to add DNA sequence into the genome of bacteria, through the transposon mechanism. Insertion of this foreign DNA would occur between EcoRI XbaI (prefix) and SpeI PstI (suffix). These sites are surrounded by Is10 sequences. This is a Universal Transposon Plasmid.

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 up to 25µg/mL, at 50 µg/mL Pseudovibrio denitrificans is sensitive to kanamycine. (Sensitivity to antibiotics)
   
   
   
2. Phenotypic verification
   
   
   
   Fig. 1 Image of Pseudovibrio denitrificans colonies in a marine broth petri dish containing kanamycin 50 µg/mL. Pseudovibrio denitrificans were previously transformed with the pNK2-CRPIIh plasmid.
   Pd corresponds to Pseudovibrio denitrificans not transformed.
   Pd1, Pd2, Pd3, Pd4, Pd5 correspond to Pseudovibrio denitrificans transformed.
   
   
3. Genotypic verification
   
   
   a) Amplification of transposon
   
   The kanamycin gene is included in the transposon sequence of pNK2 and we thus amplified this gene for our PCR verification.
   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 lambda pir cells, we assumed the amplification we obtained were from ADNg and not from plasmids.
   
   Fig. 2 Electrophoresis gel of samples coming from the PCR which amplifies the kanamycine Resistance cassette. As expected, all clones have the insertion of the transposon, except the control.
   
   
   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. 3 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, generating small fragments ready for religation.
   After the recircularization, we searched an 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 relinearized fragments we could perform a PCR to find:
   
   
   Fig. 4 Electrophoresis 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 bands of the same size, which could prefigure the existence of favored 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.
   The biobrick must be digested with bglII, extracted and ligated to form the transposon plasmid. The disgest product must migrate in the electrophoresis gel. The band to 2,6 kb, which is the biggest, corresponds to the transposon plasmid.
   To integrate any DNA sequence/biobrick into the genome, biobrick prefix and suffix can be used. This will integrate the DNA between the two transposable elements IS10. The voltage used is 2000V and the incubation time after the electroporation was 3h.
   
   
   
   Fig. 5 Electrophoresis gel of Transposon plasmid isolated from pSB1C3

   Sequence and Features

   
   

   Assembly Compatibility:

   • 10
     INCOMPATIBLE WITH RFC[10]

     Illegal prefix found in sequence at 2415
     Illegal suffix found in sequence at 2437
     
   • 12
     INCOMPATIBLE WITH RFC[12]

     Illegal EcoRI site found at 2415
     Illegal NheI site found at 947
     Illegal SpeI site found at 2438
     Illegal PstI site found at 2452
     Illegal NotI site found at 2421
     Illegal NotI site found at 2445
     
   • 21
     INCOMPATIBLE WITH RFC[21]

     Illegal EcoRI site found at 2415
     Illegal BglII site found at 5
     Illegal BglII site found at 2628
     Illegal BamHI site found at 2475
     
   • 23
     INCOMPATIBLE WITH RFC[23]

     Illegal prefix found in sequence at 2415
     Illegal suffix found in sequence at 2438
     
   • 25
     INCOMPATIBLE WITH RFC[25]

     Illegal prefix found in sequence at 2415
     Illegal XbaI site found at 2430
     Illegal SpeI site found at 2438
     Illegal PstI site found at 2452
     
   • 1000
     COMPATIBLE WITH RFC[1000]