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Team:Evry/Biology/CellCharacterization/TransformationProtocol
From 2014.igem.org
Transformation Protocol
Blabla chimio impossible
Blabla proto + schéma
Table X: Results of washing test, number of CFU for the different washes.
Blabla proto+schéma
Table Y: Results of washing test, number of CFU for the different voltage.
![Table]()
Blabla différents test+mutations envisagées
plasmides testés (rappel des collabo)
Figure Z: Maps of PSB1C3.
Blabla échec
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 M: Result of promoter sequencing.
Thus, there is 94.1% of identity between the transketolase promoter of the Pseudovibrio FOBEG1 strain ans the Pseudovibrio ascidiaceicola strain.
For the replication origin, the aim was here to find the replication origin of the plasmid of the reference strain FOBEG1. In this way, repA, repB and repC genes were looked for on NCBI. Those three genes follow each other in the genome.
This sequence was amplified with primers 49 and 50 (lien primers).
Figure Q: Result of ORI amplification in Pseudovibrio denitrificans (Pd), Pseudovribrio ascidiaceicola (Pa) and E.coli DH5a.
Figure N: Maps of PBBR1MCS and pRhoKHI-2.
Blabla final sur échec => EcoK1 DNAseq
