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Transformation Protocol

First we trying to transform Pseudovibrio denitrificansby a heat shocK. Chemo-competent cells were prepared with CaCl2. Transformation protocols were carried out by mixing 50µL of cells with 1µL of plasmid PSB1C3 solution (35ng/µL). After the standard heat shock protocol for E.coli, cells were sowed on MB 1X plates. This protocol led to no transformants.
By studying transformation of bacteria phylogenetically close of our strain of Pseudovibrio denitrificans, we noticed that marines bacteria are often resistant to common chemical transformation approaches (Piekarski T, Buchholz I, 2009) .

We envisaged to test protocols of conjugation and electroporation to transform our bacteria. Unfortunately, due to the lack of time we just tested the electroporation approach.
Two important parameters had to be finalized; the type and the number of cell washes and the voltage.

  • Washing test
    Marine bacteria live in a salty environment and a classic water wash risks to lead to an osmotic shock. Thus we are tested washes with glycerol 10% and sorbitol 2% to try to limit the shock.
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    Figure6: Protocol of washing tests

    Pre-cultures of Pseudovibrio denitrificans and E.coli were prepared and incubated overnight, respectively at 30°C and 37°C with shaking. After do the protocol (see section Protocols) of electrocompetents cells, cells were sowed 20µL of pre-culture when the DO(600nm)=1.5 for Pseudovibrio and 0.5 for E.coli on plates corresponding to the media used for pre-cultures. CFU were then counted for all plates (Table5).

    Table5: Results of washing test, number of CFU for the different washes.
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    Washes' tests reveal that sorbitol is the best way to wash cells in MB pre-culture. Gycerol can also be used. For pre-cultures in M9, the better way to wash cells is by using glycerol.

  • Voltage test
    For this part, we have to find the voltage which allows the best efficiency, by keeping living cells but by being strong enough to transform our bacteria.
    To achieve this goal, we made a similar protocol as the washes protocol described previously, by testing different voltage on our cells, without plasmids (Figure7).
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    Figure7: Protocol of voltage tests

    Pre-cultures of Pseudovibrio denitrificans and E.coli were prepared and incubated overnight, respectively at 30°C and 37°C with shaking. After do the protocol (see section Protocols) of electrocompetents cells, we electropored our cells at 1200 V, 1800 V, 2000 V and 2200 V. Cells were after incubated respectively with shaking at 30°C for Pseudovibrio and 37°C for E.coli. Then we sowed those cells on MB or M9 plates according to the environment used for the pre-culture.

    Unfortunately, some plates were convered by cellular lawn and it was impossible to count the CFU. We chose to estimate boorishly the cells' growth as shown in the Table 6.

    Table 6: Results of voltage tests.

    In this table we can see that the higher voltage for which we obtain a very good rate of living cells is 2000 V for Pseudovibrio. According to these results, wo chose to practice a 2000 V electroporation to transform our Pseudovibrio denitrificans

  • Test with PSB1C3
    Thise year, all the biobricks had to be sent in the backbone PSB1C3. For our first tries to transform Pseudovibrio denitrificans, we thus chose to test this plasmid.
    We made the competent cells protocol and then we effected the developed transformation protocol (see section Protocols), which is an electroporation at 2000 V.
    Figure8: Maps of PSB1C3.

    Blabla échec

  • Other plasmids tested
    Blabla plasmides testés (rappel des collabo)

    Figure9: Maps of PBBR1MCS and pRhoKHI-2.

  • 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 (see primers table in Protocols).

    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.

    Figure10: 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 (see primers table in Protocols).

    Figure11: Result of ORI amplification in Pseudovibrio denitrificans (Pd), Pseudovribrio ascidiaceicola (Pa) and E.coli DH5a.

    The negative control with E.coli DH5a confirms that there is no amplification. For Pseudovibrio strains, we obtain one band for Pseudovibrio denitrificans and two for Pseudovibrio ascidiaceicola.

    Meanwhile the plasmide of the transposase was tested, and the project to create a viable plasmide for pseudovibrio was abandoned (Transposons ).
    Moreover, our third hypothesis of a system which degrade exogen DNA was nourishing by BLAST data, and the presence of the enzyme EcoK1 in the genome (see section Genome assembly). This enzyne is in deed kwnon to degrade DNA without a certain pattern of methylation.