Team:Evry/Notebook/CellCharacterization

From 2014.igem.org

IGEM Evry 2014

Notebook - Cell Characterization

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New protocols of electrocompetent Pseudovibrio - Results

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The transformation didn't work with Pseudovibrio washed 5 times or 6 times.

Oct 09
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New protocols of electrocompetent Pseudovibrio
Transformation was perfomed on the new stocks made the 10-06-14 with two types of washes. 1µL of plasmids PBBR1MCS and pRhokHI-2 (25 and 33ng/mL).
2000 V
(Protocole)

Oct 07
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New protocols of electrocompetent Pseudovibrio
On a pre-culture of Pseudovibrio in MB 1X 3mL, we relaunch the culture in 100mL of MB 1X. We made the electrocompetent protocol (Protocole).
We tested to make 5 and 6 washes with glycerol 10% instead of three. Stocks of cells were stored at -80°C (3 weeks maximum)

Oct 06
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Transformation of Pseudovrbrio with pBBR1MCS and pRhokHI-2 - Confirmation
We tried to confirm the presence of our plasmid of previous transformations by extracting potential plasmids and digest them with EcoRI. After the incubation and the inactivation of the enzyme, products were migrating.
No band on the gel. The control (lab stock) didn't work either, so there is probably a problem of quantity of DNA.

Sep 17
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Transformation of Pseudovrbrio with pBBR1MCS and pRhokHI-2 - Results
The transformation didn't work.

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Stock of pBBR1MCS and pRhokHI-2 - Results
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Sep 11
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Transformation of Pseudovrbrio with pBBR1MCS and pRhokHI-2 - Confirmation
A contamination has been shown in another transformation test. We can't use our previous results because there is a chance that it's not Pseudovibrio but the contamination which had been transformed. We can still try to prove that it's our bacteria (with amplification of specific sequence) ond that there is our plasmids.

New transformation was tried with another stock of competent Pseudovibrio (Protocole) and cells were plated on MB/MB+Cam(1:1000)/MB+Kan(1:1000).

Stock of pBBR1MCS and pRhokHI-2
The colony of E.Coli transformed in LB 1X + Cam (1:1000) culture of 3mL was sowed on new LB/LB+Cam(1:1000)/LB+Kan(1:1000) plates and incubated with shaking overnight at 37°C.

Sep 10
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Transformation of Pseudovrbrio with pBBR1MCS and pRhokHI-2 - Confirmation
The previous digestion could fail because of the too little time of incubation (15min). We re-try to make the digestion with an incubation of 1 hour.

IMAGE
Gel of digestion with XbaI (2)

There was a problem with the digestion. We are going to try with another enzyme.

Stock of pBBR1MCS and pRhokHI-2
To remake a stock of these plasmids, we showed a colony of E.Coli transformed in a new LB 1X + Cam (1:1000) culture of 3mL.

Sep 09
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Transformation of Pseudovrbrio with pBBR1MCS and pRhokHI-2 - Confirmation

We made a restriction control of our plasmid, and we tried to prove that these plasmids are present in our Pseudovibrio.
After made a purification of plasmids in A1 and A2 with NucleoSpin Plamsid Kit (Macherey Nagel), we obtained concentrations at 380.1 ng/µL for pRhokHI-2 and at 91.1 ng/µL for pBBR1MCS.

We disgested these sample and our stock of pRhokHI-2 and pBBR1MCS with XbaI (unique restriction site on these plasmids) and we have make migrate the product of digestion.

IMAGE
Gel of digestion with XbaI

There was a problem with the digestion. We are going to try with a longer time of incubation.

Stock of pBBR1MCS and pRhokHI-2
To remake a stock of these plasmids, we put in liquid LB+Cam (1:1000) culture, a colony from a LB+Cam plate of E.Coli transformed.

Sep 08
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New stock of electro competents Pseudovribio - Antibiotics' control

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Sep 06
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New stock of electro competents Pseudovribio

We choose to make a new stock of electro competent Pseudovibrio denitrificans.
We made a culture of 100mL from a pre-culture of 10mL made the 04/09.
The new stock was test on plates MB 1X with Cam and Kan (1:1000).

Sep 05
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Transformation of Pseudovrbrio with pBBR1MCS and pRhokHI-2 - Results

  • Positive control with E.Coli :
    GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE

  • Resulst with Pseudovibrio :
    GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE

    NB: The negative control with E.Coli does'nt work on any plates. The culture was probably too old.

    A problem occurs with stocks of electro-competent Pseudovibrio because wa can see that it growth on Kanamycine 1:1000. On Chloramphenicol, the contamination does'nt seem to grow.
    We have to prove that it's Pseudovibrio wich growth on Chloramphenicol.

    Sep 02
  • Picture

    Transformation of Pseudovrbrio with pBBR1MCS and pRhokHI-2 - Transformation

    pBBR1MCS pRhokHI-2 Ø plasmid
    Pseudo replicate 1 Pseudo replicate 1 Pseudo
    Pseudo replicate 2 Pseudo replicate 2 E.Coli Bl21
    E.Coli Bl21 E.Coli Bl21

    Transformation of Pseudovrbrio with pBBR1MCS and pRhokHI-2 - Selection
    Plates are showed and organised like shown in the following picture:

    GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE
    A1 B1 C1 D1
    Pseudo+pBBR1MCS (1) Pseudo+pBBR1MCS (2) Pseudo Pseudo Ø plasmid
    A2 B2 C2 D2
    Pseudo+pRhokHI-2 (1) Pseudo+pRhokHI-2 (2) Pseudo Pseudo Ø plasmid



    A3 B3 C3
    E.Coli Bl21 Bl21 Ø plasmid Bl21+pRhokHI-2

    A4 B4 C4
    E.Coli Bl21 Bl21 Ø plasmid Bl21+pBBR1MCS



    We also made MB only plates to see the normal growth of our bacteria,which were showed and organised like shown in the following picture:

    GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE


    A B C D a b c d e f
    E.Coli Bl21 Bl21 Ø plasmid Bl21+pBBR1MCS Bl21+pRhokHI-2 Pseudo Pseudo Ø plasmid Pseudo+pRhokHI-2 (1) Pseudo+pRhokHI-2 (2) Pseudo+pBBR1MCS (1) Pseudo+pBBR1MCS (2)

    Sep 01
    Picture

    Transformation of Pseudovrbrio with pBBR1MCS and pRhokHI-2 - Competents cells
    New stock of electro-competent Pseudovibrio was made from a pre-culture of 100mL. (Protocole)

    Aug 29
    Picture

    Amplification of pRhokHI-2 in E.Coli Bl21 - Results
    After incubation overnight we can see some colonies of E.Coli Bl21 transformed on LB+Cam 1:1000 and on LB+Kan 1:1000. Our Bl21 which had not been transformed doesn't growth on these media but both grow on LB only. We had well success to tranform E.Coli and amplifly the plasmid.

    Transfo2


    We tranfered two colonies into liquid MB+Kan(1:1000) and let them in incubator overnight to have a pre-culture for the plasmid purification with NucleoSpin Plamsid Kit (Macherey Nagel).

    Aug 28
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    Differed launch of antibiotics' tests

  • Tests of Ampicilin's resistance cassette:
    Graph and photo of Ampicilin's resistance tests on MB 1X plates-Day3
    New Graphs with AmpR

    Graph and of Ampicilin's resistance tests on MB 0.5X plates-Day3
    New Graphs with AmpR

    Graph and of Ampicilin's resistance tests on M9 1X plates-Day3
    New Graphs with AmpR

    Photos of plates-Day3
    New Graphs with AmpR

    Aug 28
  • Picture

    Amplification of pRhokHI-2 in E.Coli Bl21
    The transformation of E.Coli with pRhokHi-2 still didn't works. We still have to amplify it so we re-tried to the tranformation (1µL of plasmid for 40µL of cells, 1800V).
    This time, after a liquid culture of only one hours cells were plated on LB 1X + Kan (1:1000), LB 1X + Cam (1:1000), and LB only and let in incubator overnight.

    Aug 27
    Picture

    Differed launch of antibiotics' tests

  • Tests of Ampicilin's resistance cassette:
    Graph of Ampicilin's resistance tests on MB 1X plates-Day2
    New Graphs with AmpR

    Graph of Ampicilin's resistance tests on MB 0.5X plates-Day2
    New Graphs with AmpR

    Graph of Ampicilin's resistance tests on M9 1X plates-Day2
    New Graphs with AmpR

    Aug 27
  • Picture

    Transformation of Pseudovribrio with pBBR1MCS - Results
    We saw no colony on our plates. We decided to test our two replicates of transformation on MB 1X plates with other concentrations of antibiotic. We made three plates with Cam 1:5000, and 1:10 000, then Pseudovibrio were showed on them and let overnight in incubator.

    Aug 26
    Picture

    Differed launch of antibiotics' tests

  • Tests of Ampicilin's resistance cassette:
    Graph of Ampicilin's resistance tests in liquid M9 1X-Day3
    New Graphs with AmpR

    Graph of Ampicilin's resistance tests on MB 1X plates-Day1
    New Graphs with AmpR

    Graph of Ampicilin's resistance tests on MB 0.5X plates-Day1
    New Graphs with AmpR

    Graph of Ampicilin's resistance tests on M9 1X plates-Day1
    No readable results

    Aug 26
  • Picture

    Amplification of pRhokHI-2 in E.Coli Bl21
    We re-tried to transform E.Coli with pRhokHI-2 to amplify it (1µL of plasmid for 40µL of cells, 1800V). This time, after a liquid culture of only one hours cells were plated on LB 1X + Kan (1:1000) and let in incubator overnight.

    Transformation of Pseudovribrio with pBBR1MCS
    After made a purification of pBBR1MCS with NucleoSpin Plamsid Kit (Macherey Nagel), electrocompetent Pseudovibrio cells were tranformed with the plasmid by electroporation (1µL of plasmid for 40µL of cells, 1800V), then they were incubated for three hours and showed on MB 1X+Cam (1:1000) plates. Plates are let in incubator overnight.

    Aug 25
    Picture

    Differed launch of antibiotics' tests

  • Tests of Kanamycin's resistance cassette:
    Graph of Kanamycin's resistance tests in liquid M9 1X-Day3
    New Graphs with KanR

  • Tests of Ampicilin's resistance cassette:
    Graph of Ampicilin's resistance tests in liquid M9 1X-Day2
    New Graphs with AmpR

    Aug 25
  • Picture

    Amplification of plasmids pBBR1MCS and pRhokHI-2 in E.Coli Bl21 - Results

    Transfo1

    Amplification of pBBR1MCS works and we purified it with NucleoSpin Plamsid Kit (Macherey Nagel) .
    Unfortunately the transformation with pRhokHI-2 didn't works so we have to retry.

    Aug 24
    Picture

    Differed launch of antibiotics' tests

  • Tests of Kanamycin's resistance cassette:
    Graph of Kanamycin's resistance tests in liquid M9 1X-Day2
    New Graphs with KanR

  • Tests of Ampicilin's resistance cassette:
    Graph of Kanamycin's resistance tests in liquid M9 1X-Day1
    New Graphs with AmpR

    Aug 24
  • Picture

    Amplification of plasmids pBBR1MCS and pRhokHI-2 in E.Coli Bl21
    Two plasmids pBBR1MCS and pRhokHI-2 were generously sent by Dr Thomas DREPPER (University Duesseldorf, Dept. of Biology, Institute of Molecular Enzyme Technology)

    plasmidS

    Bowth were transfered into Bl21 electro-competent cells by electroporation (two replicate)(1µL of plasmid for 40µL of cells, 1800V).
    After a liquid culture of three hours, cells were plated on LB 1X + Cam (1:1000) and let in incubator overnight.

    Aug 23
    Picture

    Antibiotics' tests on MB 1X plates - Results Day3
    Results of antibiotics' tests on plate are calculated as percentage of bacteria's growth by compared with the controle case. Pseudovibrio coming from culture with antibiotics (part B on plates), are taken from antibiotics' range in M9 at Day2.

  • Chloramphenicol Cam
  • Kanamycin Kan
  • Ampicilin Amp
  • Tetracyclin Tet
  • Erythromycin Erm
    Graphs
    Antibiotics' tests on MB 0.5X plates - Results Day3
    Results of antibiotics' tests on plate are calculated as percentage of bacteria's growth by compared with the controle case. Pseudovibrio coming from culture with antibiotics (part B on plates), are taken from antibiotics' range in M9 at Day2.
  • Chloramphenicol Cam
  • Kanamycin Kan
  • Ampicilin Amp
  • Tetracyclin Tet
  • Erythromycin Erm
    Graphs
    Antibiotics' tests on M9 1X plates - Results Day3

    Results of antibiotics' tests on plate are calculated as percentage of bacteria's growth by compared with the controle case. Pseudovibrio coming from culture with antibiotics (part B on plates), are taken from antibiotics' range in M9 at Day2.

  • Chloramphenicol Cam
  • Kanamycin Kan
  • Ampicilin Amp
  • Tetracyclin Tet
  • Erythromycin Erm
    Graphs


    Differed launch of antibiotics' tests
  • Tests of Kanamycin's resistance cassette:
    Graph of Kanamycine's resistance tests in liquid M9 1X-Day1
    New Graphs with KanR
  • Tests of Ampicilin's resistance cassette:

    Antibiotics' tests in liquid M9 for E.Coli transformed with a plasmid PSB1A3 containing an ampicilin resistance cassette were launch. In two days, these cultures will be plated on MB 1X, MB 0.5X and M9 1X plates

    Aug 23
  • Picture
    Antibiotics' tests on MB 1X plates - Results Day2

    Results of antibiotics' tests on plate are calculated as percentage of bacteria's growth by compared with the controle case. Pseudovibrio coming from culture with antibiotics (part B on plates), are taken from antibiotics' range in M9 at Day2.

  • Chloramphenicol Cam
  • Kanamycin Kan
  • Ampicilin Amp
  • Tetracyclin Tet
  • Erythromycin Erm
    Graphs

    photo

    Antibiotics' tests on MB 0.5X plates - Results Day2

    Results of antibiotics' tests on plate are calculated as percentage of bacteria's growth by compared with the controle case. Pseudovibrio coming from culture with antibiotics (part B on plates), are taken from antibiotics' range in M9 at Day2.

  • Chloramphenicol Cam
  • Kanamycin Kan
  • Ampicilin Amp
  • Tetracyclin Tet
  • Erythromycin Erm
    Graphs

    photo

    Antibiotics' tests on M9 1X plates - Results Day2
    Results of antibiotics' tests on plate are calculated as percentage of bacteria's growth by compared with the controle case. Pseudovibrio coming from culture with antibiotics (part B on plates), are taken from antibiotics' range in M9 at Day2.
  • Chloramphenicol Cam
  • Kanamycin Kan
  • Ampicilin Amp
  • Tetracyclin Tet
  • Erythromycin Erm
    GraphsAug 22
  • Picture

    Antibiotics' tests in liquid M9 1X - Results Day3

  • Chloramphenicol Cam
  • Kanamycin Kan
  • Ampicilin Amp
  • Tetracyclin Tet
  • Erythromycin Erm
    Graphs
    Antibiotics' tests on MB 1X plates - Results Day1
    Results of antibiotics' tests on plate are calculated as percentage of bacteria's growth by compared with the controle case. Pseudovibrio coming from culture with antibiotics (part B on plates), are taken from antibiotics' range in M9 at Day2.
  • Chloramphenicol Cam
  • Kanamycin Kan
  • Ampicilin Amp
  • Tetracyclin Tet
  • Erythromycin Erm
    Graphs

    Photos

    Antibiotics' tests on MB 0.5X plates - Results Day1
    Results of antibiotics' tests on plate are calculated as percentage of bacteria's growth by compared with the controle case. Pseudovibrio coming from culture with antibiotics (part B on plates), are taken from antibiotics' range in M9 at Day2.
  • Chloramphenicol Cam
  • Kanamycin Kan
  • Ampicilin Amp
  • Tetracyclin Tet
  • Erythromycin Erm
    Graphs

    Photos"

    Antibiotics' tests on M9 1X plates - Results Day1
    Results of antibiotics' tests on plate are calculated as percentage of bacteria's growth by compared with the controle case. Pseudovibrio coming from culture with antibiotics (part B on plates), are taken from antibiotics' range in M9 at Day2.
  • Chloramphenicol: No readable results
  • Kanamycin: No readable results
  • Ampicilin: No readable results
  • Tetracyclin: No readable results
  • Erythromycin: No readable results

    Aug 21
  • Picture

    Antibiotics' tests in liquid M9 1X - Results Day2

  • Chloramphenicol Cam
  • Kanamycin Kan
  • Ampicilin Amp
  • Tetracyclin Tet
  • Erythromycin Erm
    Graphs
    Launch of antibiotics' tests on plates
    Plates are showed and organised like shown in the following picture, and showed with 1µL of each culture:
    GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE
    A B C D
    Pseudovibrio denitrificans Pseudovribrio denitrificans* E. Coli E. Coli**
    *Bacteria cultivated in media with antibiotic
    **Bacteria tranformed with a plasmid containing a resistance cassette for the antibiotic

    Aug 20
  • Picture

    Antibiotics' tests in liquid M9 1X - Results Day1

    • Chloramphenicol GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE
    • Kanamycin GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE Because of technical problems, the tests on E.Coli+KanR are launched with a gap of few days.
    • Ampicilin GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE
    • Tetracyclin GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE
    • Erythromycin GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE
      GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE

      Aug 19
    Picture

    Tests of antibiotics' stocks - Results(2)

    Plates LB Agar only LB+Kan LB+Erm (1:100)
    E.coli X 0 0

    New stocks are ready for antibiotics' tests on Pseudovribrio

    Launch of antibiotics's tests in liquid M9 1X
    For each tested antibiotic we made the following range of concentrations:

    0 1:500 1:1000 1:50 000 1:100 000

    We made these range two time to have a replicate and we added a controle tube without antibiotic for each bacteria. Each tube is made like in the following table in Falcon tubes (15mL):


    / 0 1:500 1:1000 1:50 000 1:100 000
    Medium+agar 3mL 3mL 3mL 3mL 3mL
    Antibiotic 0 6µL of stock 3µL of stock 6µL of stock diluted 1/100 3µL of stock diluted 1/100
    Pre-culture 300µL 300µL 300µL 300µL 300µL

    For each range, we test Pseudovibrio denitrificans but also E.Coli and E.Coli tranformed with a speicific resistance cassette (NM:The E.Coli strain for the control case is the same strain that is tranformed for the cassette efficiency test). The initial OD at 600nm of each bacteria has been mesured to after be able to calculate the ΔOD at 600nm, during three days.


    / Pseudo Bl21 DH5a Top 10 DH5a pyr+KanR DH5a+CamR Top 10+ErmR
    OD init 0.02 0.795 0.873 0.693 0.408 0.687 0.319

    Plates for antibiotics's tests
    For each tested antibiotic we made the same range of concentrations than in liquid. We also made these range two time to have a replicate and we added a controle plate without antibiotic on which we showed all our tested bacteria. Plates will be checked during three days. We made these same ranges of tested antibiotic's concentrations for three media:

    • MB agar 1X
    • MB agar 0.5X
    • M9 agar 1X

    Each plate is made like in the following table:


    / 0 1:500 1:1000 1:50 000 1:100 000
    Medium+agar 20mL 20mL 20mL 20mL 20mL
    Antibiotic 0 40µL of stock 20µL of stock 40µL of stock diluted 1/100 20µL of stock diluted 1/100

    Aug 18
    Picture
    Tests of antibiotics' stocks - Results

    Plates LB Agar only LB+Cam LB+Kan LB+Amp LB+Erm LB+Tet
    E.coli X 0 X 0 X 0

    There was a problem with the stock of Kanamycine so we made a new one at 25mg/mL. For the erythrompycine, we learn that E. Coli is not really sensitive to this antibiotic and that it's better to use it with a dilution at 1:100.


    Survivability tests - Results

    liquid cultures/Plates MB M9
    MB ++++ ++
    M9 ++ +

    Any liquid culture can be plated and will grow up. Of course, it works better on MB medium, wich is a rich medium than on M9.

    Aug 17
    Picture

    Tests of antibiotics' stocks
    Six plates of LB agar were made. Five of them contained one of those antibiotics in the dilution 1:1000:

  • Chloramphénicol
  • Kanamycin
  • Erythromycin
  • Ampicilin
  • Tetracyclin
    (The last one was the control of the growth of our bacteria without antibiotics) We sowed 10µL of a liquid LB culture of Bl21, and we let them incubate overnight at 37°C.

    Survivability tests
    Two plates of MB 1X and M9 1X were made and divised in two parts. Each plate was sowed with 5µL of a liquid MB 1X culture of Pseudovibrio denitrificans(dated 12th August) in a part and with 5µL of a liquid M9 1X culture Pseudovibrio denitrificans (dated 12th August) in the other part.

    Pre-cultures
    Cultures in MB 1X and M9 1X of Pseudovibrio were passed by a 1/10 dilution and let incubate overnight at 30°C. New cultures of different E.Coli were also started from glycerol or plate and let incubated overnight at 37°C.

    From glycerol stocks:
  • Bl21
  • Top10
  • DH5a

    From plates:
  • DH5a tranformed with pCB1C3 (CamR)
  • Top10 transformed with pQexp (ErmR)
  • DH5a pyr tranformed with pMK2 (KanR)

    Bacteria tranformed with plasmid were cultivated with the corresponding antibiotic for the selection. For each medium we make a negative contrôle without bacteria.

    Aug 16
  • Picture

    Tests for development of the electroporation's protocol
    *Results of M9 1X plates - Day2:

    POUIT


    Cold sorbitol seems to be the best way to wash our Pseudovibrio cells and avoid cells' death on M9 plates too.



    *Results CFU:
    POUIT

    Cold sorbitol is the best way to wash cells because it induce the bigest number of colonies on MB1X and bigger colonies on M91X.



    Aug 13
    Picture

    Tests for development of the electroporation's protocol

    *Results of M9 1X plates - Day1:

    POUIT


    Results on M9 plates are not very readable.



    *Results of MB 1X plates - Day1:

    POUIT


    Cold sorbitol seems to be the best way to wash our Pseudovibrio cells and avoid cells' death on MB plates.

    Aug 12
    Picture

    Tests for development of the electroporation's protocol
    *Showed of MB Pseudovribrio electro-competent (which are stocked at -80°C) on MB 1X plates.
    *Showed of M9 Pseudovribrio electro-competent (which are stocked at -80°C) on M9 1X plates.

    Aug 11
    Picture

    Tests for development of the electroporation's protocol
    Because of technical problems, we have to re-try with a culture of Pseudvirbrio in MB 1X.
    Same protocol as 5th August.

    Aug 06
    Picture

    Tests for development of the electroporation's protocol

  • Re-launch of 50mL of Pseudovribrio in MB and M9 from pre-culture of 4th August.
  • Incubation at 30°C and waiting for the exponential growth phase.
    > OD(600nm) in M9 = 0.15
    > OD(600nm) in MB = 1.5

    WORK IN ICE

  • Centrifuge 10min at 4000rmp and 4°C
  • Wash cells 1:1 of the initial volume with cold sorbitol, cold MiliQ H2O or cold glycerol 10%
  • Centrifuge 10min at 4000rmp and 4°C
  • Wash cells 1:2 of the initial volume with cold sorbitol, cold MiliQ H2O or cold glycerol 10%
  • Centrifuge 10min at 4000rmp and 4°C
  • Wash cells 1:10 of the initial volume with cold sorbitol, cold MiliQ H2O or cold glycerol 10%
  • Centrifuge 10min at 4000rmp and 4°C
  • Resuspend cells 1:100 of the initial volume with cold sorbitol, cold MiliQ H2O or cold glycerol 10%
  • Stock cells in aliquots of 50µL at -80°C

    Aug 05
  • Picture

    Tests for development of the electroporation's protocol
    *Pre-culture of Pseudovibrio denitrificans in 3mL of Marine broth 1X
    *Pre-culture of Pseudovibrio denitrificans in 3mL of M9+CasAmino Acids 1X

    Incubation overnight, 30°C.

    Aug 04

    Picture

    Voltage tests
    text to print if image not found

    July 31
    Picture

    Curve of growth
    We previously tried to af a curve of grotwh by mesuring the DO (600nm), but because of the opaqueness of the MB these data are useless.
    We measured the growth of Pseudovibrio denitrificans on the TECAN in M9-CASA+3%NaCl and MB filtred (because of the opaqueness of the non filtred MB)

    Curve
    Curve2

    Unfortunately, cells didn't grow very well in the TECAN due to the little volume use and the ratio air/culture which is not optimal.

    July 30
    Picture

    Construction of a new plasmid - Choice and amplification of the ORI
    Purification of these PCR products.
    Use “GenJEt” PCR Purification for it. The protocol is same that PCR purification except that the binding buffer is add (1μL for 1mg) and boil at 55°C during 10 mn.
    After the purification, a new PCR is realized with 3μL to purificated fragments.
    Send to sequencing.

    July 25
    Picture

    Construction of a new plasmid - Choice and amplification of the ORI
    PCR with

  • primers 49 and 50 (see primers table in Protocols)
  • Q5 high fidelity enzyme
  • TM=60°C.

    Migration of the PCR products, we have two bands per well.

    Figure
    Figure: 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.


    July 23
  • Picture

    Construction of a new plasmid - Choice and amplification of the ORI
    We chose to amplify the genes RepA, RepB and RepC. We find these three genes with the genome browser on the genome of Pseudovibrio FOBEG1.
    PCR with

  • primers 49 and 50 (see primers table in Protocols)
  • Q5 high fidelity enzyme
  • TM=55°C.

    Migration of the PCR products, we have no band the amplification didn't work.

    July 22
  • Picture

    Construction of a new plasmid - Verification of the promoter
    A PCR was performed with primers 5 and 6 (see primers table in Protocols).
    We purified those PCR product and sent them to be sequenced
    Results :

    Figure


    July 21
    Picture

    Construction of a new plasmid - Choice and amplification of the promoter
    We chose to amplify the tkt promoter of the transkelotase gene. We find this strong constitutive promoter with the genome browser on the genome of Pseudovibrio FOBEG1.
    PCR with primers 5 and 6 (see primers table in Protocols), Q5 high fidelity enzyme and TM=55°C.

    July 18
    Picture

    Transformation of Pseudovibrio with PSB1C3 (2) - Results
    We have no colonies on the plates.

    July 16
    Picture

    Transformation of Pseudovibrio with PSB1C3 (2)
    We made electrocompetent Pseudovibrio and transform them by electroporation with

  • 1µL of palsmid (33ng/µL)
  • 50µL of cells
  • 2000V and sowed cells on MB 1X+Cam 1/1000 after 2hours of incubation.

    July 15
  • Picture

    Transformation of Pseudovibrio with PSB1C3 - Results
    We have no colonies on the plates.

    July 13
    Picture

    Transformation of Pseudovibrio with PSB1C3
    We made electrocompetent Pseudovibrio and transform them by electroporation with

  • 1µL of palsmid (33ng/µL)
  • 50µL of cells
  • 1800V and sowed cells on MB 1X+Cam 1/1000 after 2hours of incubation.

    July 12
  • Picture

    Curve of growth
    We try to measure a curve of grotwh by mesuring the DO (600nm) of a culture of Pseuovibrio denitrificans in MB 1X with a spectrometer

    Curve
    Unfortunately these data looks like useless. Because of the opaqueness of the media, reliable data are not possibles to obtain.

    July 10