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Team:Evry/Notebook/CellCharacterization
From 2014.igem.org
Notebook - Cell Characterization
New protocols of electrocompetent Pseudovibrio - Results
The transformation didn't work with Pseudovibrio washed 5 times or 6 times.
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)
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)
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.
Transformation of Pseudovrbrio with pBBR1MCS and pRhokHI-2 - Results
The transformation didn't work.
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.
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.
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.
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.
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.
New stock of electro competents Pseudovribio - Antibiotics' control
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).
Transformation of Pseudovrbrio with pBBR1MCS and pRhokHI-2 - Results
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
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:
| 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:
| 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
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)
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.
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
Differed launch of antibiotics' tests
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.
Differed launch of antibiotics' tests
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.
Differed launch of antibiotics' tests
No readable results
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.
Differed launch of antibiotics' tests
Aug 25
Amplification of plasmids pBBR1MCS and pRhokHI-2 in E.Coli Bl21 - Results
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.
Differed launch of antibiotics' tests
Aug 24
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)
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.
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.
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.
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.
Differed launch of antibiotics' tests
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
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.
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.
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.
Aug 22
Antibiotics' tests in liquid M9 1X - 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.
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.
"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.
Antibiotics' tests in liquid M9 1X - Results Day2
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 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
| 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:
(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:
From plates:
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.
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.
Tests for development of the electroporation's protocol
> OD(600nm) in M9 = 0.15
> OD(600nm) in MB = 1.5
WORK IN ICE
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.
Voltage tests
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.
Construction of a new plasmid - Choice and amplification of the ORI
PCR with
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
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.
Transformation of Pseudovibrio with PSB1C3 (2) - Results
We have no colonies on the plates.
Transformation of Pseudovibrio with PSB1C3 (2)
We made electrocompetent Pseudovibrio and transform them by electroporation with
July 15
Transformation of Pseudovibrio with PSB1C3 - Results
We have no colonies on the plates.
Transformation of Pseudovibrio with PSB1C3
We made electrocompetent Pseudovibrio and transform them by electroporation with
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
July 10
