Difference between revisions of "Team:Pasteur Paris/Test"

07.16.2018

We transformed pET 43.1a and pSB1C3 in DH5-α competent cells, in order to constitute a stock of empty vectors for our manipulation.

Plasmid	C (ng/μl)	Volume (μl)	Competent cell	Medium
pET 43.1.a	4.95	1	DH5-α	LB/carbenicilline
pSB1C3	40	1	DH5-α	LB/chloramphenicol

See here the transformation of E. coli DH5-alpha protocol
We let the transformed bacteria grow overnight (16 hours).

07.17.2018

Results:
We went to see our bacterial culture:

• Bacteria transformed with pET 43.1 had not grown.
• Bacteria transformed with pSB1C3 had formed colonies.

Interpretations:
Bacteria transformed with pET 43.1 did not grow. We found a non-commercial tube of pET 43.1 in the freezer from last year team, and we decided to try to amplify it because we did not have any commercial tubes of pET 43.1. Transformation did not work as expected, probably because:

• There was no DNA left in the tube.
• The concentration of DNA was too low .

We cultivated the transformed pSB1C3 bacteria in liquid medium 2 x 25ml + Chloramphenicol (25µg/ml) overnight at 37°C, 180 rpm.

Results:
Bacteria successfully transformed with pSB1C3.
(See here the liquid culture protocol)

07.18.2018

Extraction:
We extracted the pSB1C3 plasmid from the bacterial culture.
The protocol used was the Qiagen Plasmid Purification Kit (See Midiprep for plasmid extraction protocol here )
Measure of the DNA concentration in each tube thanks to the NanoDrop (Blank used : TE.1) (See the NanoDrop protocol here)

Results:
We used the Nanodrop to quantify the purified DNA.

Sample	1	2	3	4	5	6	7	8	9	10
Volume (µl)	50	50	50	50	50	50	50	50	50	50
C (ng/µl)	-7.8	-8.5	54.8	5.6	237.4	-7.9	-7.2	-7.5	-6.9	-6.5
Abs(260 nm)/Abs(280 nm)	1.43	1.42	1.97	3.04	1.85	1.32	1.47	1.56	1.35	1.44

Remarks:
[NA] < 0 = no DNA
[NA] > 200 good
Abs_{(260 nm)}/Abs_{(280 nm) [1;2]}

Interpretations:

• Tube 1, 2, 6, 7, 8, 9 and 10 did not contain DNA at all. This problem can be due to the fact that we failed the precipitation with ethanol, meaning that when we removed all the supernatant, we also removed the DNA, or that we removed by mistake the DNA pellet when removing all the supernatant after precipitation (the pellet was so small that we couldn’t see it with the naked eye).
• Tube 3, 4 and 5 had DNA inside. The amount of DNA obtained is enough for us. These tubes have been placed in the freezer for later use.

Transformation:
We received from Eurofins our first sequences:

• 3a_NGF construction Part1 (Seq1)
• 3a_NGF construction Part2 (Seq2)
• T7 RIP construction (Seq8)

In order to constitute a stock of this commercial vectors, we transformed DH5-α competent cells with the plasmid we received, and then we cultivated them on agar plates covered with LB medium and carbenicillin.
As the bacteria transformed with pET 43.1a did not grow, we tried again to transform the bacteria with pET 43.1a in a higher quantity than previously.

Plasmid	C (ng/μl)	Volume (μl)	Competent cell	Medium
pEX-A258 Seq1	10	1	DH5-α	SOC/carbenicilline
pEX-A258 Seq2	10	1	DH5-α	SOC/carbenicilline
pEX-A258 Seq8	10	1	DH5-α	SOC/carbenicilline
pET 43.1a	4.95	5	DH5-α	SOC/carbenicilline

See here the transformation of E. coli DH5-alpha protocol

07.19.2018

Results:
We went to see our overnight bacterial culture plates:

	SEQ#1 3a_NGF_Part1	SEQ#2 3a_NGF_Part2	SEQ#8 T7_RIP	Pet43.1a(+)
Selection	Amp	Amp	Amp	Amp
Growth	Yes	Yes	Yes	No
Liquid Culture	Yes	Yes	Yes	No

Interpretations:
Transformation of Seq#1 – 3a_NGF_Part1, Seq#2 – 3a_NGF_Part2 and Seq #8 – T7_RIP did work as expected.
Transformation of Pet43.1a (+) did not work again. As mentioned before, the tube of pET43.1a (+) was a non-commercial tube that we found in the freezer from last year’s team, and we decided to try to amplify it because we did not have any commercial tubes of pET 43.1. We tried to transform our bacteria twice, with no results, so the most probable hypothesis is that the tube did not contain DNA anymore. After this experiment, we decided to buy a tube pET43.1a(+).

We cultivated two different colonies from each plate (seq#1, seq#2, seq#8):

• One in an Erlenmeyer in 25 ml LB medium + Carbenicillin (100µg/ml) overnight at 37°C, 180 rpm.
• One in two Falcon tube in 15 ml LB medium + Carbenicillin (100µg/ml) overnight at 37°C, 180 rpm.

(See here the liquid culture protocol)

07.20.2018

Results of the liquid culture are good.

Extraction - MidiPrep:
We extracted the 3 plasmids Seq#1 – 3a_NGF_Part1, Seq#2 – 3a_NGF_Part2 and Seq #8 – T7_RIP from the bacterial cultures.
The protocol used was the Qiagen Plasmid Purification Kit (See Midiprep for plasmid extraction protocol here )
Measure of the DNA concentration in each tube thanks to the NanoDrop (Blank used : TE.1) (See the NanoDrop protocol here)

Results:
We used the Nanodrop to quantify the purified DNA.

For NGF part1, Colony 1:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
3569.2	1.9
1578.7	1.89
848.1	1.90
2322.1	1.89

For NGF part1, Colony 2:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
488.1	1.89
493.7	1.90
445.1	1.89
644.6	1.86

For NGF part2, Colony 1:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
1555.0	1.88
493.7	1.88
666.2	1.87
367	1.90

For NGF part2, Colony 2:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
463.7	1.89
420.8	1.88
461.4	1.89
420.6	1.89

For RIP, Colony 1:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
694.4	1.84
1028.9	1.87
1408.7	1.88
466.8	1.88

For RIP, Colony 2:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
228.4	1.89
325.2	1.89
379.4	1.89
427.2	1.89

Remarks:
[NA] < 0 = no DNA
[NA] > 200 good
Abs_{(260 nm)}/Abs_{(280 nm) [1;2]}

Interpretations:
We have DNA in our tubes. The DNA is too concentrated so we will have to dilute them before next use. We froze the tubes at -20°C until next use.

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07.24.2018

Culture of strain HB2151 transformed with pVDL 9.3 (received on 23.07.2018 from Dr. Victor de Lorenzo)

Material:

• LB agar plate supplemented with chloramphenicol (25 mg/mL)
• Bacteria HB 2151 transformed with pVDL9.3

The bacteria we received was in the form of a stock: LB + agar media containing the bacteria and the plasmid in an Eppendorf tube. Sample from the solid stock was streaked on LB agar plate complemented with chloramphenicol, and secondly grown in an Erlenmeyer with 25 mL LB medium and 25 μL chloramphenicol at 25 mg/mL.

Biofilm culture on filter (0.2 μm)

Material:

• Bacteria DH5α transformed with pSB1C3
• Filters - 0.2 μm Milipore (5 cm diameter)
• Petri-dishes
• Sterile Clamps
• Sterile Rakes
• Pipets
• LB media
• Falcon tubes
• Chloramphenicol (25 mg/mL)

1. Make a liquid culture of DH5α transformed with pSB1C3 from colonies we had on a petri dish made on the 07.16.2017 and stored at 4°C. Let grow over night at 37°C under agitation.
   Made the 07.23.2017
   (See here the liquid culture protocol)
2. Auto-cleavage of the 0.2 μm filters
3. Deposit the filters on a petri dish with a clamp. Wait until the filter is totally “wet” with LB media.
4. Deposit the bacteria culture in the center of the filter with a micropipette and spread with a rake. We made 3 different petri dishes with 3 different volumes.

	DH5α-pSB1C3- biofilm culture – 1	DH5α-pSB1C3- biofilm culture – 2	DH5α-pSB1C3- biofilm culture – 3
Volume of culture deposit (μL)	50	100	150

5. Incubate at 37°C overnight.

Transformation of seq5, seq7 from Eurofins Genomics, pET 43.1 and pBR322
Material:

• LB agar plate supplemented with carbenicillin
• Sterile LB medium
• pET 43.1a: 42.36 pg/μL
• pBR322: 65.00 ng/μL
• Seq5 > AGR-RIP Part1: 1.1 μg
• Seq7 > AGR-RIP Part3: 1.9 μg

Sequences received on the 07.24.2018.
Gene synthesis products are in the form of lyophilized powder, so we need to re-suspend them.

Seq5 > AGR-RIP Part1: 1.1 μg
Seq7 > AGR-RIP Part3: 1.9 μg

1. Centrifuge in order to make sure that all the plasmids are at the bottom of the tube and not in the lid.
2. Re-suspend the powder in TE 1X.
   Seq5: add 110 μL to have a final concentration of 10 ng/μL
   Seq7: add 190 μL to have a final concentration of 10 ng/μL
3. Transformation of the 4 plasmids in DH5α competent cells:
   • Seq5, Seq7 and pBR322: 1 μL
   • pET 43.1a: 10 μL
   
   (See here the transformation of E. coli DH5-alpha protocol)
4. Culture on petri dishes overnight at 37°C

07.25.2018

Transformed bacteria cultures:
We went to see our overnight bacterial culture plates:

	Seq5 RIP_Part1	Seq7 RIP_Part3	pBR322	Pet43.1a(+)
Selection	Amp	Amp	Amp	Amp
Growth	Yes	Yes	Yes	No
CFU	2 816	2 492	2 628	-
Liquid Culture	Yes	Yes	Yes	No

Interpretations:
Transformation of Seq5 – RIP_Part1, Seq7 – RIP_Part3 and pBR322 did work as expected.
Transformation of Pet43.1a (+) did not work again. The tube of pET43.1a (+) was another non-commercial tube that we found in the freezer from last year’s team, and we decided to try to amplify it because we did not have any commercial tubes of pET 43.1. We tried to transform our bacteria twice, with no results, so the most probable hypothesis is that the tube did not contain DNA anymore.
We are still waiting for the commercial pET43.1a(+) (we ordered it last week).

Bacteria transformed pVDL 9.3:
The culture on agar plate did work, but we were not able to isolate one colony.
We stopped the liquid culture and measured the OD600 of the culture medium.

Biofilm culture:

	pSB1C3-DH5α-biofilm culture – 1	pSB1C3-DH5α-biofilm culture – 2	pSB1C3-DH5α-biofilm culture – 3
Volume of culture deposit (μL)	50	100	150
Biofilm formation	Yes	Yes	Yes

We did a biofilm transfer. We took our filter with the biofilm and transferred it on another porous filter placed on a new petri dish LB + Cm. Then we removed the filter containing the biofilm and incubated the plate overnight at 37°C.

Liquid culture:
We cultivated two different colonies from each plate (Seq5, Seq7 and pBR322):

• One in an Erlenmeyer in 25 mL LB medium + Carbenicillin (100 µg/ml) overnight at 37°C, 180 rpm.
• One in two Falcon tube in 15 mL LB medium + Carbenicillin (100 µg/ml) overnight at 37°C, 180 rpm.

Moreover, for each colony of each DNA part we did some streaks on a plate and incubated them at 37°C. (See here the liquid culture protocol)

Midiprep pVDL 9.3:
We extracted the pVDL 9.3 plasmid from the HB2151 strain.
The protocol use was the Qiagen Plasmid Purification Kit (See Midiprep for plasmid extraction protocol here )
We divided the eluted DNA into 8 tubes before centrifugation.

Measure of the DNA concentration in each tube by the NanoDrop.
Blank used : TE.1 (See the NanoDrop protocol here)

Results:
We used the Nanodrop to quantify the purified DNA for SEQ: pVDL 9.3 1.

Sample	1	2	3	4	5	6	7	8
ng/µL [NA]	3.7	111.2	140	0	1.9	47	108	8.3
Abs(260 nm)/Abs(280 nm)	1.93	1.87	1.88	-	1.6	1.9	1.9	1.91

07.26.2018

Results:
Transfer of our biofilm growth on a filter (07.24.2018 loading of 50 µl of pSB1C3 liquid culture on a filter) on another filter on a plate and incubation overnight at 37°C:

Observations:
The biofilm is able to grow again after a transfer from one filter to another as seen by the red color.

Midiprep Seq5, Seq7, pBR322:
We extracted Seq5, Seq7 and pBR322 plasmid from transformed DH5α strain.
The protocol used was the Qiagen Plasmid Purification Kit (See Midiprep for plasmid extraction protocol here )
We divided the eluted DNA into 4 tubes before centrifugation.

Measure of the DNA concentration in each tube with NanoDrop.
Blank used: TE.1 (See the NanoDrop protocol here)

Results:
We used the Nanodrop to quantify the purified DNA.

For Seq, 5 Colony 1:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
245.7	1.87
349	1.87
353.2	1.87
306.7	1.87

For Seq 5, Colony 2:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
258	1.88
281	1.86
260	1.86
295	1.86

For Seq 7, Colony 1:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
293	1.88
299	1.88
305	1.87
288	1.87

For Seq 7, Colony 2:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
217	1.87
212	1.87
175	1.88
244	1.83

For pBR322, Colony 1:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
3.8	1.68
55.8	1.86
54	1.9
68	1.84

For RIP, Colony 2:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
32	1.82
28	1.75
41	1.88
30	1.88

Interpretations:
There is DNA in all the tubes. For Seq5 and Seq7, we obtain high DNA concentrations, which is good. For pBR322, the DNA concentration is lower. This is probably due to the fact that pB322 is a low copy plasmid. However, the quantity of DNA is enough.

Preparing cultures for stocks:
We set some pre-cultures of Seq5, Seq7 and pBR322, in order to make some stocks to store at -80°C. In a 15 mL Falcon tube, we placed 5 mL of LB media, and 10 μL of antibiotic (Carbenicillin). We added one of each colony inside each Falcon and placed the Falcon tubes inside the incubator (37°C – 180 rpm) and let grow overnight.

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