Antibiotics' tests in liquid M9 1X - Results Day3

Chloramphenicol

Kanamycin

Ampicilin

Tetracyclin

Erythromycin

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

Kanamycin

Ampicilin

Tetracyclin

Erythromycin

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

Kanamycin

Ampicilin

Tetracyclin

Erythromycin

"

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

Antibiotics' tests in liquid M9 1X - Results Day2

Chloramphenicol

Kanamycin

Ampicilin

Tetracyclin

Erythromycin

Launch of antibiotics' tests on plates
Plates are showed and organised like shown in the following picture, and showed with 1µL of each culture:

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

Antibiotics' tests in liquid M9 1X - Results Day1

• Chloramphenicol
• Kanamycin Because of technical problems, the tests on E.Coli+KanR are launched with a gap of few days.
• Ampicilin
• Tetracyclin
• Erythromycin
  
  Aug 19

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

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

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

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

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

*Results CFU:

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

Tests for development of the electroporation's protocol

*Results of M9 1X plates - Day1:

Results on M9 plates are not very readable.

*Results of MB 1X plates - Day1:

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

Aug 12

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

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

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

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

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)

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

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

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: 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

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

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 :

July 21

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

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

July 16

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

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

July 13

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

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

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

July 10