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Team:Evry/Biology/CellCharacterization/TransformationProtocol
From 2014.igem.org
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) .
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.
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 Protocoles) 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).
Voltage test
Blabla proto+schéma
Test with PSB1C3
Blabla différents test+mutations envisagées
plasmides testés (rappel des collabo)
Figure7: Maps of PSB1C3.
Blabla échec
Other plasmids tested
Figure8: 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.
Figure9: 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).
Figure10: 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 DNAseq in Cell Charactherizaion ). This enzyne is in deed kwnon to degrade DNA withour a certain pattern of methylation.
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.
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.
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 Protocoles) 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.
Blabla proto+schéma
Table 6: Results of voltage test, number of CFU for the different voltage.
Blabla différents test+mutations envisagées
plasmides testés (rappel des collabo)
Figure7: Maps of PSB1C3.
Blabla échec
Figure8: Maps of PBBR1MCS and pRhoKHI-2.
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.
Figure9: 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).
Figure10: 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 DNAseq in Cell Charactherizaion ). This enzyne is in deed kwnon to degrade DNA withour a certain pattern of methylation.
