Team:Evry/Biology/CellCharacterization/TransformationProtocol

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Transformation Protocol




Blabla chimio impossible


  • Washing test
    Blabla proto + schéma
    Table X: Results of washing test, number of CFU for the different washes.
    text to print if image not found


  • Voltage test
    Blabla proto+schéma
    Table Y: Results of washing test, number of CFU for the different voltage.
    Table


  • Test with PSB1C3
    Blabla différents test+mutations envisagées
    plasmides testés (rappel des collabo)
    Figure
    Figure Z: Maps of PSB1C3.

    Blabla échec

  • Create a new plasmid
    After trying to transform Pseudovibrio denitrificans with the PSB1C3 whic didn't work, we had several hypothesis:
  • The promoter of the Chloramphenicol resistance gene doesn't work in our bacteria
  • The origin of replication doesn't work in our bacteria
  • There is a system degrading exogene DNA in Pseudovibrio
    To check these hypothesis, we chose to find specific constitutive promoter and origin of replication in another member of the genus Pseudovibrio, which has been sequenced, the FOBEG1 strain. (lien article)

  • A strong constitutive promoter of FOBEG1 was looked for with the genome browser. Upstream sequences of vital and cell cycle independant genes were explored. We were interested by the transkelotase, key enzyme of the way of pentoses phosphates.
    We can suspect that this enzyme has to have a good constitutive promoter, or at least led a reliable and consequent expression. To be sure to have the whole promoter sequence, we exported the sequence (fasta format) from the beginning of the ORF of the transkelotase, until the end of the previous ORF. This sequence was amplified with primers 5 and 6 (lien primer).

    After a PCR, we obtained a 6000pb band for Pseudovibrio ascidiaceicola, and no band for Pseuvibrio denitrificans. These PCR product was sequenced, as show in figure M.

    Figure
    Figure M: Result of promoter sequencing.


  • Other plasmids tested

    Figure
    Figure N: Maps of PBBR1MCS and pRhoKHI-2.

    Blabla final sur échec => EcoK1 DNAseq

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