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

07.10.2018

Institut Curie’s lab MMBM (Macromolécules et Microsystèmes en Biologie et en Médecine) at IPGG (Institut Pierre Gilles de Gennes) handed us the molds for the microchannel chip and granted us access to their lab. We have been taught the basic steps for the fabrication of the microfluidic microchannel chip: PDMS preparation, molding, demolding, punching, bonding.

Basic steps protocols here .

07.11.2018

4 microchannel chips have been made. Molds for the PDMS well chip have been received. Glass microscopic slides coated with 2 nm chromium and 50 nm gold have been received for use in PDMS well chip. Glass slides have been cut into small strips with a diamond tip. Processing damaged the gold coating.

Microchannel chip fabrication protocols here .

07.12.2018

First pieces of the well chip have been successfully molded. 8 microchannel chips have been made.

Microchannel chip fabrication protocol here .

Well chip fabrication protocol here .

07.13.2018

Tested the conductivity of gold coated strips of glass. Current doesn't seem to pass through the strips. It is supposed that the numerous scratches caused by the cutting process are the cause for the non-conductivity of the strips. It was realized after the cutting process, that the conductivity should also have been measured before cutting.

Second successful set of PDMS pieces of the well chip molded. 4 microchannel chips have been made. 1 microchannel successfully bonded to an imaging dish, and filled with fluorescein. Observation under a microscope revealed that it is fully functional.

Bonding protocol here .

Microchannel chip protocol here .

Well chip fabrication protocol here .

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:
[DNA] < 0 = no DNA
[DNA] > 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:

[DNA] (ng/µL)	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 [DNA]	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 [DNA]	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 [DNA]	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 [DNA]	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.

Something was done one that day

18.07.2018

1 set of PDMS pieces for the well chip has been made. One of the pieces broke while demolding. It was discarded. 4 microchannel chips made.

Well chip fabrication protocol here .

Microchannel chip fabrication protocol here .

19.07.2018

1 set of PDMS pieces for the well chip have been made. 4 microchannel chips made.

Well chip fabrication protocol here .

Microchannel chip fabrication protocol here .

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.

Something was done one that day

07.24.2018

Membrane filters from Sterlitechreceived. 8 microchannel chips and 1 set of PDMS pieces for the well chip have been made.

Well chip fabrication protocol here .

Microchannel chip fabrication protocol here .

07.25.2018

Platinum wires received and conductivity tested. Function generator was set on sine. The physical quantities measured here are Eg, the generator's tension amplitude and Ep, the voltage difference between the two extremities of a platinum wire. the quantity calculated here is 20*log(Ep/Eg) for different frequencies.

Results

Interpretation

Voltage difference calculated is extremely low, indicating a very good conductivity for the platinum wires, so its resistance (in low frequencies) can be neglected at first glance when it will be used in PDMS well chips. Resistance increases in higher frequencies, because of the skin-effect in metals: the strip transforms into an antenna. But as we are going to use only low frequencies, this doesn't affect us.

Conductivity measurement protocol (section 2, replace chip by platinum wire, no resistor) here .

07.27.2018

4 microchannel chips have been made. 1 microchannel chip bonded for imaging test purposes.

Microchannel chip fabrication and bonding protocol here .

07.30.2018

Digestion of Seq8 and pBR322:
We cut the commercial plasmid containing our Seq8 (T7-RIP) with restriction enzymes XbaI and SpeI, and pBR322 with AvaI and BsmI, in order then to insert our sequence into the plasmid pBR322.
Material:

• Water baths at 37°C and 80°C
• Eppendorf tubes
• Pipets and cones

Reagents:

• pBR322 [68 ng/μL]
• commercial plasmid containing Seq8 [466,8 ng/μL]
• Restriction enzymes: AvaI, BsmI, XbaI, SpeI
• 10X Cut Smart
• Distilled water

1. Mix the following volumes of reagents and incubate at 37°C for 15 minutes.

	Volume (μL)
pBR322	22
10X Cut Smart	5
Distilled water	20
Bsml	1.5
Aval	1.5

	Volume (μL)
Eurofins plasmid with Seq8	20
10X Cut Smart	5
Distilled water	22
Xbal	1.5
Spel	1.5

2. Inactivate the digestion by heating the mixture in a heat block at 80 °C for 20 minutes.

 

Electrophoresis on agarose gel

We prepared an Agarose gel.
(See here the Agarose Gel Preparation protocol)

We filled:

• 1 well with the digested pBR322 (n°1)
• 2 wells with the digested plasmids containing Seq8 (n°2 and 3)
• 1 well with a DNA ladder (n°4)

We let the DNA migrate for 10 minutes at 85 V and then 40 minutes at 125 V.
We then revealed the gel under UV lamp.

Results:

Interpretations:

• pBR322 (4361 bp) has been successfully linearized
• pEXA258 from Eurofins Genomics has also been successfully digested. We obtained two fragments corresponding to our insert (459 bp) and the linearized plasmid (2909 bp)

DNA Gel extraction:
Here is the same gel after DNA extraction of pBR322 and of Seq8.

Sequence name	Eppendorf weight (g)	Eppendorf + gel weights (g)	Gel weight (g)
Seq8 (1)	1.08	1.29	0.21
Seq8 (2)	1.08	1.37	0.29
pBR322	1.08	1.27	0.19

It is useful to know how the weight of the gel to adapt de volumes of buffer to the amount of gel we have.
(See here the Gel Extraction protocol)
Results: after extracting the DNA from the gel, we measured the DNA concentration using the Nanodrop.

Sequence name	DNA concentration (ng/μL))	Abs(260nm)/Abs(280nm) (g)	Total extracted DNA (ng)
Seq8 (1)	6.4	1.87	294.4 (620.8)
Seq8 (2)	6.8	2.07	326.4 (620.8)
Linearized pBR322	9.9	1.97	475.2

Interpretations:
The initial amount of each DNA fragment before digestion was 1500 ng.
We can therefore evaluate the efficiency of our digestion and extraction:

• for Seq8 insert: r = 0.414
• for pBR322 plasmid: r = 0.317

Ligation of linearized pBR322 and our insert Seq8

We used the In-Fusion cloning kit provided by Ozyme in order to clone our insert into the linearized pBR322.

1. We mixed the following volumes into 1.5 mL tubes.

Component	Cloning (μL)	Negative control (μL)	Positive control (μL)
Purified fragment	4	-	2 (control)
Linearized vector	4	1	1 (control)
5X In-Fusion enzyme premix	2	2	2
Deionized water	-	7	5

2. Incubate for 15 min in a water bath at 50°C
3. Ligation See here the Ligation protocol)
4. Set in ice
5. Transformation of the 3 plasmids into Stellar competent cells (provided with the cloning kit)
   • Competent cell volume : 33 μL
   • DNA volume: 2 μL
   (See here the transformation of E. coli DH5-alpha protocol)
6. Culture on petri dishes LB agar gel supplemented with carbenicillin overnight at 37°C

Transformation of Seq6 from Eurofins
Material:

• LB agar plates supplemented with carbenicillin (25 μg/mL)
• LB medium
• Seq6 > AGR-RIP Part1: 4.6 μg

Sequences are under the form of lyophilized powder, so we need to re-suspend it.

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.
   We resuspended it into 460 μL TE, to obtain a concentration of 10 ng/μL.
3. Transformation of the plasmid in DH5α competent cells
   (See here the transformation of E. coli DH5-alpha protocol)
4. Culture on petri dishes LB agar gel supplemented with carbenicillin overnight at 37°C

07.31.2018

Results: ligation cultures

As expected, we could observe a lot of colonies for the positive control. Some colonies had grown for the negative control (due to the linearized plasmid which circularized itself). For our ligated plasmid, we only observed a few colony (as much as in the negative control) so we didn’t know if the ligation succeeded. This is why we picked up 20 colonies and cultivated them overnight, hoping that one of these had the correct plasmid.

The plate containing DH5-α transformed with Seq6 also had a lot of colonies, as expected.

Liquid culture of DH5-α with Seq6
We cultivated 2 different colonies in 250 mL Erlenmeyer containing:

• 25 mL LB medium
• 25 μL carbenicillin (25 mg/mL)

We cultivated it overnight in a shaking incubator at 37°C, 180 rpm.
(See here the liquid culture protocol)

Liquid culture of Stellar transformed with pBR322-Seq8

We cultivated 20 different colonies in order to maximize our chance to get one successful ligated plasmid. Each colony was cultivated in a 15 mL Falcon tube containing:

• 5 mL LB medium
• 5 μL carbenicillin (25 mg/mL)

We cultivated it overnight in a shaking incubator at 37°C, 180 rpm.
We also cultivated each colony on petri dishes LB agar plate supplemented with carbenicillin overnight at 37°C.

Transformation of Seq3 from Eurofins Genomics and commercial pET43.1a
Material:

• LB agar plate supplemented with carbenicillin
• Seq3 : NGF-GFP Part1 : 4.0 μg
• pET 43.1a : 0.5 μg/μL

Sequence from Eurofins Genomics under the form of lyophilized powder so we need to re-suspend it.

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. We resuspended it into 400 μL TE, in order to obtain a concentration of 10 ng/μL.
3. Transformation of the plasmids in DH5α competent cells:
   • volume of competent cells: 50 μL
   • pET 43.1a: 50 pg (1 μL of commercial plasmid diluted 1/10000)
   • Seq3 : 10 ng (1 μL)
   (See here the transformation of E. coli DH5-alpha protocol)
4. Culture on petri dishes LB agar plate supplemented with carbenicillin overnight at 37°C.

08.01.2018

We went to see our cultures:

• All the colonies of our ligation (pBR322 + Seq8) grew except one.
• DH5α transformed with Seq3 grew.
• DH5α transformed with pET 43.1a did not grow.

Hypothesis:

• We might have diluted the plasmid or the bacteria too much.
• We did not homogenize the solution enough during the dilution of the commercial solution.
• We did not pipet correctly the 1 μL of plasmid from the commercial tube.

 

Extraction of the ligated plasmid (pBR322 + Seq8) from Stellar (miniprep)
(See here the Gel Extraction protocol)
Results: (tube 8 did not grow)
From NanoDrop

Sample	DNA CONCENTRATION (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	94.8	2.05
2	105.8	2.06
3	106.3	2.04
4	84.8	2.02
5	100.1	2.02
6	79.9	2.00
7	08.9	2.04
8	-	-
9	115.1	2.02
10	116.7	1.96
11	76.6	2.00
12	69.5	1.99
13	80.7	1.94
14	63.0	1.98
15	78.4	2.03
16	153.1	1.94
17	90.7	2.00
18	119.3	2.02
19	100.8	1.97
20	85.3	2.03

We chose to verify which colony had acquired the plasmid containing the Seq8 insert. To do so, we digested the plasmid with the restriction enzymes EcoRI and PstI and ran a 1% agarose gel.
The size of the plasmid pBR322 is 4361 bp. Once we cut the sequence between the sites EcoRI (bp position 4359) and PstI (bp position 3611), this leaves us with an empty backbone of 3613 bp. The size of the insert Seq8 is 461 bp.
Here we made a mistake, by confusing the right restriction enzymes to use. Indeed, we should have used XbaI and SpeI instead of EcoRI and PstI. Moreover, as these two restriction sites are present in the pBR322 backbone (748 bp away), this digestion should show two DNA fragments.

1% Agarose gel of the miniprep of the ligated plasmid (pBR322 + Seq8) from Stellar
Only the first half of the samples (1 to 11) were deposed today.
The preparation of the gel was done according to the Agarose Gel Preparation protocol.
The samples were made according to the following proportions:

• 5 µL DNA
• 3 µL 10X Cutsmart Buffer
• 1 µL EcoRI
• 1 µL PstI
• 20 µL H2O

These samples were then incubated for 10 min at 37°C to allow digestion by the enzymes.
The reaction was stopped by the addition of 6 µL of 6X Loading Dye.
Results:

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This resulting gel shows the two DNA fragments we expected. Moreover, the column 3 shows a lower molecular weight (MW) than others for the plasmid part. As this part is supposed to contain our insert, it might suggest that colony 3 doesn’t possess the insert, but all others do.
However, we will need to make the right experiment using restriction enzymes XbaI and SpeI (see date 08.02.2018) to verify that our insert was correctly put into the plasmid.

Extraction of Seq6 plasmid from DH5α (midiprep)

We extracted the plasmids containing Seq6 from the DH5-α cells.
(See Midiprep for plasmid extraction protocol here )

Results from NanoDrop: (Blank: TE.1)

For the colony 1:

	ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
1	1572.9	1.88
2	2800.1	1.88
3	2598.7	1.88
4	2285.1	1.89

For the colony 2:

	ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
1	4996.8	1.87
2	3263.2	1.89
3	3625.0	1.90
4	2339.7	1.90

The DNA concentrations were too high and so did not answer to the Beer-Lambert’s law. We needed to dilute the samples before using the nanodrop again. We did 1/10 dilutions and obtained average concentrations of 34 ng/µL, with Abs_{(260 nm)}/Abs_{(280 nm)} ratios of 1.92.

Digestion of pET 43.1a, NGF_Part1 (Seq1) and NGF_Part2 (Seq2)

Digestion of the following plasmids:

• pET43.1a (500 ng/µl)
• Commercial plasmid containing Seq1 (445.1 ng/μL)
• Commercial plasmid containing Seq2 (420.6 ng/μL)
• Restriction enzymes: NdeI, PacI, NheI, BamHI, MscI, HindIII

1. Mix the following volumes of reagents and incubate at 37°C for 15 min.

	Volume (μL)
pET43.1a	1.6
10X Cut Smart	5
Distilled water	40.4
Ndell	1.5
Pacl	1.5

	Volume (μL)
Eurofins plasmid with Seq1	4.3
10X Cut Smart	5
Distilled water	37.7
Nhel	1.5
BamHI	1.5

	Volume (μL)
Eurofins plasmid with Seq2	4.4
10X Cut Smart	5
Distilled water	37.6
Mscl	1.5
MindIII	1.5

2. Inactivate the digestion by heating the mixture in a heat block at 80 °C for 20 min.

Electrophoresis on agarose gel

We prepared an 0.8 % agarose gel to separate the different fragments of the cut plasmids.
(See here the Agarose Gel Preparation protocol)

We filled:

• 2 wells with digested pET43.1a (n°1 and n°2)
• 2 wells with insert Seq1 (n°3 and n°4)
• 2 wells with insert Seq2 (n°5 and n°6)
• 1 well with the DNA ladder

We let the DNA migrate for 10 minutes at 85 V on order to let the DNA penetrate the gel, and then 40 min at 125 V to efficiently separate the fragments.
We then revealed the gel under UV lamp.

Interpretations:

• pET43.1a (7275 bp) has been successfully linearized into two fragments (5 kb and 2 kb)
• pEXA258 – Seq1 from Eurofins Genomics has also been successfully digested. We obtained two fragments corresponding to our insert (1105 bp) and the linearized plasmid (2446 bp)
• pEXA258 – Seq2 from Eurofins Genomics has also been successfully digested. We obtained two fragments corresponding to our insert (1159 bp) and the linearized plasmid (2446 bp)

DNA Gel extraction:
We cut the gel to extract our inserts and linearized plasmid:

	Eppendorf weight (g)	Eppendorf + gel weights (g)	Gel weight (g)
pET43.1a (1)	1.13	1.365	0.23
pET43.1a (2)	1.08	1.286	0.21
Seq1 (1)	1.07	1.265	0.20
Seq1 (2)	1.08	1.306	0.23
Seq2 (1)	1.07	1.303	0.23
Seq2 (2)	1.07	1.284	0.21

It is useful to know how the weight of the gel to adapt de volumes of buffer to the amount of gel we have.
(See here the Gel Extraction protocol)

Results: After extracting the DNA from the gel, we measured the DNA concentration using the Nanodrop.

	C (ng/μL)	Abs(260 nm)/Abs(280 nm)
pET43.1a (+) (1)	5.5	3.14
pET43.1a (+) (2))	7.1	2.50
Seq1 (1)	5.1	2.71
Seq1 (2)	7.8	1.90
Seq2 (1)	6.9	2.92
Seq2 (2)	6.0	3.11

Ligation of linearized pET43.1a and our inserts Seq1,2 /br>
We used the In-Fusion cloning kit provided by Ozyme to clone our two inserts together in linearized pET43.1a.

1. We mixed the following volumes into 1.5 mL tubes.

Component	Cloning (μL)	Negative control (μL)	Positive control (μL)
Purified Seq1	6	-	2 (control)
Purified Seq2	6	-	2 (control)
Linearized vector	3	1	1 (control)
5X In-Fusion enzyme premix	4	2	2
Deionized water	1	7	5

2. Incubate for 15 min in a water bath at 50°C
3. Ligation (See Ligation protocol)
4. Set on ice
5. Transformation of the 3 plasmids into Stellar competent cells (provided with the cloning kit):
   • Competent cell volume: 50 μL
   • DNA volume: 2 μL
   (See here the transformation of E. coli DH5-alpha protocol)


6. Culture on petri dishes LB agar plate supplemented with carbenicillin overnight at 37°C

08.02.2018

2nd Agarose gel of the miniprep of the ligated plasmid (pBR322 + Seq8) from Stellar, using the correct enzymes
Yesterday we made a mistake, so today we carried out exactly the same protocol but with restriction enzymes SpeI and XbaI. As a negative control, we also deposited a sample we called 1x, without digesting it with restriction enzymes.

Liquid culture of pET43 1.a
We cultivated 2 different colonies in 250 mL Erlenmeyer containing:

• 25 mL LB medium
• 25 μL carbenicillin (25 mg/mL)

We cultivated it overnight in a shaking incubator at 37°C, 180 rpm.
(See here Liquid Culture protocol)

Liquid culture of Stellar transformed with pET43 1.a-Seq1-Seq2
We cultivated 20 different colonies in order to maximize our chance to get one successful ligated plasmid. Each colony was cultivated in a 15 mL Falcon tube containing:

• 5 mL LB medium
• 5 μL carbenicillin (25 mg/mL)

We cultivated it overnight in a shaking incubator at 37°C, 180 rpm.
< We also cultivated each colony in an LB agar plate supplemented with carbenicillin.

Extraction of Seq3 plasmid from Stellar (midiprep)
We extracted the plasmids containing Seq3 from the stellar cells
(See here the Midiprep for plasmid extraction protocol)

Results from NanoDrop (with Blank: TE.1)
For the colony 1:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
153.3	1.90
155.2	1.90
164.6	1.89
179.1	1.91

For the colony 2:

ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
166.4	1.91
150.4	1.90
177.1	1.91
188.6	1.89

Interpretations: There is DNA in all the tubes

Digestion of pBR322, Agr-RIP-Part1 (Seq5), Agr-RIP-Part2 (Seq6) and Agr-RIP-Part3 (Seq7)

Material:

• Water baths at 37°C and heating block of 80°C
• Eppendorf tubes
• Pipets and cones

Reagents:

• PBR322 (conc. 68 ng/μL)
• Eurofins Genomics plasmid containing Seq5 (conc. 353 ng/ μL)
• Eurofins Genomics plasmid containing Seq6 (conc. 350 ng/ μL)
• Eurofins Genomics plasmid containing Seq7 (conc. 305 ng/ μL)
• Restriction enzymes: BsmI, AvaI, XbaI, BamHI, MscI, SphI, HindIII, SpeI
• 10X Cut Smart
• Distilled water

1. Mix the following volumes of reagents and incubate at 37°C for 15 min

	Volume (μL)
pBR322	22
10X Cut Smart	5
Distilled water	20
BsmI	1.5
Aval	1.5

	Volume (μL)
Plasmid containing Seq5	12
10X Cut Smart	5
Distilled water	30
Xbal	1.5
BamHI	1.5

	Volume (μL)
Plasmid containing Seq7	21
10X Cut Smart	5
Distilled water	21
HindIIIl	1.5
Spel	1.5

	Volume (μL)
Plasmid	16
10X Cut Smart	5
Distilled water	26
Mscl	1.5
Sphl	1.5

2. Inactivate the digestion by heating the mixture in a heat block at 80 °C for 20 min.

Electrophoresis on agarose gel

We prepared an 0.8 % agarose gel to separate the different fragments of the cut plasmids.
(See here the Agarose Gel Preparation protocol)

We filled:

• 2 wells with digested pBR322 (n°1 and n°2)
• 2 wells with insert Seq5 (n°3 and n°4)
• 2 wells with insert Seq6 (n°5 and n°6)
• 2 wells with insert Seq7 (n°7 and n°8)
• 1 well with the DNA ladder

We let the DNA migrate for 10 minutes at 85 V on order to let the DNA penetrate the gel, and then 40 min at 125 V to efficiently separate the fragments.
We then revealed the gel under UV lamp.

• 1. 2. : pBR322
• 3. 4. : Seq5
• 5. 6. : Seq6
• 7. 8. : Seq7

Interpretations:

• pET43.1a (7275 bp) has been successfully linearized into two fragments
• pEXA258 – Seq5 from Eurofins Genomics has also been successfully digested. We obtained two fragments corresponding to our insert (1422 bp) and the linearized plasmid (2.5 kb)
• pEXA258 – Seq6 from Eurofins Genomics has also been successfully digested. We obtained two fragments corresponding to our insert (960 bp) and the linearized plasmid (2.5 kb)
• pEXA258 – Seq7 from Eurofins Genomics has also been successfully digested. We obtained two fragments corresponding to our insert (762 bp) and the linearized plasmid (2.5 kb)

DNA Gel extraction
We cut the gel to extract our inserts and linearized plasmid:








We extract the DNA (our linearized vector and our plasmid) from the agarose gel.
(See here the Gel Extraction protocol)



Results: After extracting the DNA from the gel, we measured the DNA concentration using the Nanodrop

	ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
pBR322	10.3	2.07
Seq5	8.3	3.07
Seq6	14.0	2.06
Seq7	7.6	1.97




Ligation of linearized pBR322 and our insert Seq5,6,7

We used the In-Fusion cloning kit provided by Ozyme (See here theLigation protocol) in order to clone our insert into the linearized pBR322.


1. We mixed the following volumes into 1.5 mL tubes.

Component	Cloning (μL)	Negative control (μL)	Positive control (μL)
Purified Seq 5	5	-	2 (control)
Purified Seq 6	3	-	2 (control)
Purified Seq 7	5	-	2 (control)
Linearized vector	3	1	1 (control)
5X In-Fusion enzyme premix	4	2	2
Deionized water	-	7	5







2. Incubate for 15 min in a water bath at 50°C
3. Ligation See here the Ligation protocol)
4. Set in ice
5. Transformation of the 3 plasmids into Stellar competent cells (provided with the cloning kit)
   • Competent cell volume : 33 μL
   • DNA volume: 2 μL
   (See here the transformation of E. coli DH5-alpha protocol)
6. Culture on petri dishes LB agar gel supplemented with carbenicillin overnight at 37°C




08.03.2018





Midiprep pET43.1a:
(See here the Midiprep for plasmid extraction protocol)

2 colonies from pET43.1a petri dish (08.01.2018 – pET43.1a) were placed in liquid culture the day before

Results from NanoDrop (with Blank: TE.1)
For the colony 1:


ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
71.7	1.88
75.2	1.88
73.6	1.84
70.2	1.87

For the colony 2:


ng/µL [NA]	Abs(260 nm)/Abs(280 nm)
65.0	1.85
34.7	1.80
64.9	1.86
55.5	1.82




Observations: Enough concentrations for further uses






Miniprep pET 43.1a + Seq1 + Seq2
(See here the Miniprep for plasmid extraction protocol)
20 colonies from pET43.1a + Seq1 + Seq2 from petri dish (08.01.2018) were placed in liquid culture the day before (5 mL of LB + 10 µL of Carbenicillin).

Results from NanoDrop (Blank used is Buffer EB):

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	170.9	1.86
2	120.8	1.88
3	73.1	1.87
4	89.8	1.84
5	75.4	1.97
6	136.6	1.87
7	101.4	1.88
8	99.0	1.91
9	72.7	1.88
10	172.8	1.87
11	107.8	1.86
12	151.1	1.86
13	92.6	1.92
14	78.7	1.93
15	101.2	1.87
16	223.9	1.97
17	145.5	1.85
18	149.8	1.86
19	68.1	1.89
20	87.8	1.93




Observations: Samples were not transferred to the micro tubes at the right moment, so it is very probable that the samples are contaminated with genomic DNA.





08.05.2018





Liquid culture of pBR322 + Seq5 + Seq6 + Seq7
(See here the Liquid Culture protocol)
6 colonies from the petri dish (pBR322 + Seq5,6,7 – 08.02.2018) were placed in a liquid culture containing:

• 5 mL of LB
• 10 µL of Carbenicillin (25 mg/mL)


Cultures were incubated at 37°C, 180 rpm overnight.

Observations: All the colonies grew.

Something was done one that day

07.30.2018

First PDMS well chip entirely assembled and bonded, with platinum wire and gold membrane filter. Leaking problems required us to seal the leaks with additional PDMS. 4 microchannel chips and 1 set of PDMS pieces for the well chip have been made.

Well chip fabrication protocol (the chip assembled on that day is slightly different but doesn't work) here .

Microchannel chip fabrication protocol here .

07.31.2018

2 additional PDMS wells assembled with gold-coated membranes. First PDMS vertical chip PDMS pieces prepared (cut and punched, silver paste applied and membrane filter glued). 4 microchannel chips and 1 set of PDMS pieces for the well chip have been made.

Well chip fabrication protocol (the chip assembled on that day is slightly different but doesn't work) here .

Vertical chip fabrication protocol here .

Microchannel chip fabrication protocol here .

08.01.2018

Conductivity tests on membranes assembled on chips. Well chips not functional: tension is applied on top of the membrane, but no current flow is detected. We conclude from that, that the current can't pass from one side of the gold-coated membrane to the other. It was decided for the well chips to stop using silver paste for connecting the platinum wire to the membrane and instead mount the wire on top of the membrane.

Bonding of the vertical chip PDMS pieces failed.

Well chip conductivity protocol (section 2) here .

Vertical chip fabrication protocol here .

08.06.2018

Miniprep pBR322 + Seq5 + Seq6 + Seq7
(See here the Miniprep for plasmid extraction protocol)

Results from NanoDrop (Blank used is buffer EB):

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	61.2	2.00
2	86.3	2.03
3	58.8	1.98
4	65.9	2.00
5	74.5	2.03
6	84.4	2.07

Observations: Enough concentrations for further use.

Digestion of pET43.1a + Seq1 + Seq2

Aim: Check that the ligation has successfully worked.
Restriction enzymes used: NdeI and XbaI

Expected results:

pET43.1a	linearized	7.3 kB
pET43.1a + Seq1	1 fragment	6.63 kB
pET43.1a + Seq2	2 fragments	6.63 kB + 0.1 kB
pET43.1a + Seq1+ Seq2	2 fragments	6.63 kB + 1 kB

We digested 10 samples of our cloning products, and we also did some controls:

• Colony 10 with no enzyme (well 11)
• Plasmid pET43.1a + enzyme (well 12)
• Plasmid pET43.1a + no enzyme (well 13)

Well 1 to 10: samples from colony 1 to 10
Well 14: ladder

Procedure:

	Volume (μL)
DNA	5
Cut Smart	3
Ndel	1
Xbal	1
H2O	20

• Digestion at 37°C for 15 min
• Addition of 6 µL of loading dye

We deposited all the samples in an agarose gel previously made.
We let the gel run for 10 min at 85 V + 80 min at 125 V.

Results:

Observations: For samples 1 to 11, we can only see DNA streaks. In wells 12 and 13, we can clearly see the plasmid pET43.1a linearized (well 12) and circular (well 13). In the well 14 we can clearly see the ladder.

Interpretations: The streaks observed on the gel are probably due to the genomic DNA contamination. As mentioned the 08.03.2018, there was an error in the Miniprep of pET43.1a + Seq1 + Seq2.

Digestion of pBR322+ Seq5 + Seq6 + Seq7

Aim: Check that the ligation has successfully worked.
Restriction enzyme used: EcoRI

Expected results:

pBR322	linearized	4 kB
pBR322 + Seq5	1 fragment	5.4 kB
pBR322 + Seq5 + Seq6	2 fragments	6.3 kB
pBR322 + Seq5 + Seq6 + Seq7	2 fragments	7 kB

Procedure:

	Volume (μL)
DNA	5
Cut Smart	3
EcoRI	1
H2O	20

• Digestion at 37°C for 15 min
• Addition of 6 µL of loading dye

We digested 6 samples of our cloning products, and we also did some controls.

• Colony 6 with no enzyme (well 7)
• Plasmid pBR322 + enzyme (well 8)
• Plasmid pBR322 + no enzyme (well 9)

Wells 1 to 6: samples from colony 1 to 6
Well 10: ladder

We deposited all the samples in an agarose gel previously made.
We let the gel run for 10 min at 85 V + 90 min at 125 V.
Results:

Observations: The 6 samples are linearized and migrated exactly like the plasmid pBR322 + Enzyme. Moreover, sample 6 without enzyme migrated like pBR322 without enzyme.

Interpretations: This leads to the conclusion that none of the colonies were transformed with the plasmid AND the insert.

Liquid culture of pET43.1a + Seq1 + Seq2
(See here the liquid culture protocol)

8 colonies from the petri dish (pET43.1a + Seq1,2 from 08.01.2018) were placed in a liquid culture containing:

• 5 mL of LB
• 5 µL of Carbenicillin (25 mg/mL)

Cultures were incubated at 37°C, 180 rpm overnight.

Cloning pBR322 + Seq8
We used the In-Fusion cloning kit provided by Ozyme
in order to clone our insert in the linearized pET 43.1a.

1. We mixed the following volumes into 1.5mL tubes.

Component	Cloning (μL)	Negative control (μL)	Positive control (μL)
Purified Seq8	4	-	2 (control)
Linearized vector	15	1	1 (control)
5X In-Fusion enzyme premix	4	2	2
Deionized water	-	7	5

2. Incubate for 15 minutes in a water bath at 50°C
3. Ligation (See Ligation protocol)
4. Set in ice
5. Transformation of the 3 plasmids into Stellar competent cells (provided with the clonage kit):
   • Competent cell volume: 50 μL
   • DNA volume: 2 μL
6. Culture on petri dishes LB agar plate supplemented with carbenicillin overnight at 37°C

08.07.2018

<+br>

Miniprep pET43.1a + Seq 1 + Seq 2
(See here the Miniprep for plasmid extraction protocol)

Results from NanoDrop (Blank used is buffer EB):

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	141.2	1.84
2	173.3	1.86
3	155.2	1.83
4	151.9	1.85
5	106.9	1.85
6	143.0	1.88
7	151.1	1.89

Observations: Enough concentrations for further use.

Digestion of pET43.1a + Seq1 + Seq2

Aim: Check that the ligation has successfully worked.
Restriction enzymes used: NdeI and XbaI

Expected results:

pET43.1a	linearized	7.3 kB
pET43.1a + Seq1	1 fragment	6.63 kB
pET43.1a + Seq2	2 fragments	6.63 kB + 0.1 kB
pET43.1a + Seq1+ Seq2	2 fragments	6.63 kB + 1 kB

We digested 10 samples of our cloning products, and we also did some controls:

• Colony 8 with no enzyme (well 8)
• Plasmid pET43.1a + enzyme (well 9)
• Plasmid pET43.1a + no enzyme (well 10)

Well 1 to 7: samples from colony 1 to 8
Well 11: ladder

Procedure:

	Volume (μL)
DNA	5
Cut Smart	3
Ndel	1
Xbal	1
H2O	20

• Digestion at 37°C for 15 min
• Addition of 6 µL of loading dye

We deposited all the samples in an agarose gel previously made.
We let the gel run for 10 min at 85 V + 80 min at 125 V.

Liquid culture of pBR322 + Seq8

(See here the liquid culture protocol)

We cultivated:

• 8 colonies of Stellar bacteria transformed with the result of our cloning
• 2 colonies from the negative control

Each colony is cultivated in a 15 mL Falcon tube with 5 mL LB medium and 5 μL carbenicillin.
In parallel, we set each colony to grow on a LB agar plate supplemented with carbenicillin overnight at 37°C.

08.08.2018

Miniprep pBR322 + Seq8
(See here the Miniprep for plasmid extraction protocol)

Results from NanoDrop (Blank used is buffer EB):

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	93.0	1.97
2	101.8	1.83
3	121.7	1.98
4	112.1	2.00
5	101.9	1.98
6	97.5	1.98
7	113.4	1.98
8	97.0	2.02
Control (-) 1	91.9	2.01
Control (-) 2	73.5	1.90

Observations: Concentrations are good for further use.

Digestion of BR322 + Seq8

Aim: Check that the ligation has successfully worked.
Restriction enzymes used: NdeI

Expected results:

pET43.1a	linearized	4 kB
pET43.1a + Seq8	2 fragments	4 kB + 0.9 kB

We digested 8 samples of our cloning products, and we also did some controls:

• Colony 8 with no enzyme (well 8)
• Two colonies from the negative control of the cloning with enzyme

We filled the wells with:

• Plasmid extracted from colonies 1 to 8 and digested with NdeI (n°1 to n°8)
• Plasmid extracted from the negative control digested with NdeI (n°9 and n°10)
• Plasmid extracted from colony 8 without enzyme (n°11)

Results:

Interpretations: We observed only one fragment for all the digested plasmids, and all the fragments have the same molecular weight as the negative control (vector without insert).
Cloning did not work again.

We assume that the failure might be:

• A too low initial amount of linearized vector before cloning, resulting in all the Stellar bacteria being transformed with an empty and uncut vector.
• Too long overlaps, or too GC rich overlaps.

Digestion of pBR322, PET 43.1, AND PLASMIDS CONTAINING Seq1, Seq2 and Seq8

We plan to try cloning again with longer digestion and longer electrophoresis migration, in order to digest all the vector and to increase the efficiency of our separation.

Reagents:

• pBR322 (conc. 54 ng/μl)
• pET 43.1a (conc. 65 ng/μl)
• Seq1 (conc. 445 ng/μl)
• Seq2 (conc. 460 ng/μl)
• Seq8 (conc. 379 ng/μl)

We digest the 5 plasmids by mixing the following amounts:

	Volume (μL)
DNA	22
Cut Smart	3.2
Enzyme 1	1
Enzyme 2	1

DNA	Enzyme 1	Enzyme 2
pET 43.1a	Ndel	Pacl
pBR322	Aval	Bsml
Seq 1	Nhal	BamHI
Seq 2	Mscl	HindIII
Seq 8	Xbal	Spel

Incubation time at 37°C : 1 hour
Inactivation time at 80°C : 20 min

After digestion, we added 8 μL 6X Loading dye to each tube.

Electrophoresis on agarose gel

< We prepared a 0.8% agarose gel in order to separate the different fragments of the cut plasmids.
(See here the Agarose Gel Preparation protocol)

We filled:

• 2 wells with digested pBR322 (n°1 and n°2)
• 2 wells with digested pET 43.1a (n°3 and n°4)
• 2 wells with insert Seq1 (n°5 and n°6)
• 2 wells with insert Seq2 (n°7 and n°8)
• 2 wells with insert Seq8 (n°9 and n°10)

We let the DNA migrate for 10 min at 85 V on order to let the DNA penetrate the gel, and then 80 min at 125 V to efficiently separate the fragments. We then revealed the gel under UV lamp.

Results: DNA Gel extraction

We cut the gel to extract our inserts and linearized plasmid:
(See here the Gel Extraction protocol)

Results: After extracting the DNA from the gel, we measured the DNA concentration using the Nanodrop.

	Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
pBR322	7.0	3.85
pET43.1a	5.1	3.40
Seq1	5.7	3.40
Seq2	6.3	10.55
Seq8	5.0	4.48

Interpretations: Values of the ratio Abs_{(260 nm)}/Abs_{(280 nm)} are very high, indicating that our DNA is highly contaminated. Moreover, the concentrations obtained are too low for further use for cloning.
We have several hypotheses to explain why our experiment did not work:

• The Gel extraction kit we used might be expired (particularly the final buffer with which we elute DNA might have turned unusable)
• We need to check the wavelength of the nanodrop

We are going to try the digestion and extraction again tomorrow in order to try having better concentrations before proceeding to cloning.

08.09.2018

The digestion and extraction of yesterday did not work well (the obtained concentrations of plasmid and insert were still too low to proceed to the cloning step with a suitable volume).
Today, we will proceed again with the digestion, extraction and then try the cloning again, using this time the right amount of insert and vector (as recommended by Clontech molar ratio tool) and following the pieces of advice given to us by Takara Clontech technician we called.

Digestion of pET 43.1a, pBR322, Seq1, Seq2 and Seq8

Material:

• Water baths at 37°C and heating block of 80°C
• Eppendorf tubes
• Pipets and cones

Reagents:

• pET 43.1a (conc. 74.1 ng/μL)
• pBR322 (conc. 29.6 ng/μL)
• Eurofins Genomics plasmid containing Seq1 (conc. 1430.1 ng/ μL)
• Eurofins Genomics plasmid containing Seq2 (conc. 780.6 ng/ μL)
• Eurofins Genomics plasmid containing Seq8 (conc. 714.6 ng/ μL)
• Restriction enzymes : NdeI, PacI, NheI, BamHI, MscI, HindIII, BsmI, AvaI, SpeI, XbaI
• 10X Cut Smart
• Distilled water

1. Mix the following volumes of reagents and incubate at 37°C for 20 min

	Volume (μL)
pET 43.1a	42
10X Cut Smart	5
Distilled water	-
Nhel	1.5
Pacl	1.5

	Volume (μL)
pBR322	42
10X Cut Smart	5
Distilled water	-
Bsml	1.5
Aval	1.5

	Volume (μL)
Seq1	4.5
10X Cut Smart	5
Distilled water	31.5
Nhel	1.5
BamHI	1.5

	Volume (μL)
Seq2	8
10X Cut Smart	5
Distilled water	32
Mscl	1.5
HindIII	1.5

	Volume (μL)
Seq8	17.8
10X Cut Smart	5
Distilled water	24.2
Xbal	1.5
Spel	1.5

2. Inactivate the digestion by heating the mixture in a water bath at 80 °C for 20 min

Electrophoresis on agarose gel

We prepared a 0.8 % agar gel in order to separate the different fragments of the cut plasmids.
(See here the Agarose Gel Preparation protocol)

We filled:

• 2 wells with digested pBR322 (n°1 and n°2)
• 2 wells with digested pET 43.1a (n°3 and n°4)
• 2 wells with insert Seq1 (n°5 and n°6)
• 2 wells with insert Seq2 (n°7 and n°8)
• 2 wells with insert Seq8 (n°9 and n°10)

We let the DNA migrate for 10 min at 85 V on order to let the DNA penetrate the gel, and then 80 min at 125 V to efficiently separate the fragments.
We then revealed the gel under UV lamp.
Results of the agarose gel after migration:

Interpretations:

• pBR322 has been linearized, and we observe one fragment corresponding to the linearized plasmid
• pET 43.1a (7275 bp) has been successfully linearized into two fragments (5 kb and 2 kb)
• pEX A258 – Seq1 from Eurofins Genomics has also been successfully digested. We obtained two fragments corresponding to our insert (1105 bp) and the linearized plasmid (2446 bp)
• pEX A258 – Seq2 from Eurofins Genomics has also been successfully digested. We obtained two fragments corresponding to our insert (1159 bp) and the linearized plasmid (2446 bp)
• pEX A258 – Seq8 from Eurofins Genomics has also been successfully digested. We obtained two fragments corresponding to our insert (459 bp) and the linearized plasmid (2446 bp)

(See here the Gel Extraction protocol)

Results:
After extracting the DNA from the gel, we measured the DNA concentration using the Nanodrop

	Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
pBR322	11.1	2.19
pET43.1a	17.0	2.00
Seq1	16.9	2.19
Seq2	14.8	2.61
Seq8	15.7	2.17

Cloning :

We used the In-Fusion cloning kit provided by Ozyme in order to clone:

• Our two inserts Seq1 and Seq2 together in linearized pET 43.1a.
• Our insert Seq8 into linearized pBR322

1. Following the advice of the Takara Clontech technician we contacted, we first incubated the linearized vectors and inserts at 50°C for 5 min
2. We then placed it on ice for 3 min
3. We mixed the following volumes into 1.5 mL tubes.

Component	Cloning (μL)	Negative control (μL)	Positive control (μL)
Purified Seq1	5	-	2 (control)
Purified Seq2	6	-	2 (control)
Linearized vector	9	9	1 (control)
5X In-Fusion enzyme premix	4	2	2
Deionized water	-	9	5

Component	Cloning (μL)	Negative control (μL)
Purified Seq8	4.5	-
Linearized vector	13.5	13.5
5X In-Fusion enzyme premix	4	2
Deionized water	-	4.5

4. Incubate for 15 minutes in a water bath at 50°C.
5. Set in ice.
6. Ligation (See here the Ligation protocol)
7. Transformation of the 5 plasmids into Stellar competent cells (provided with the cloning kit):
   • Competent cell volume: 40 μL
   • DNA volume: 2 μL
   (See here the Transformation of E. coli DH5-alpha protocol)
8. Culture on LB agar plate supplemented with carbenicillin overnight at 37°C (with 1/5 dilution)

08.10.2018

Observation overnight plate culture of pBR322 + Seq8 and pET43.1a + Seq1 + Seq2 after InFusion

	pBR322 Neg Control	pBR322 + Seq8	pET43.1 Neg Control	pET43.1 + Seq1 + Seq2
CFU	46	136	27	59

Liquid culture of pBR322 + Seq8 and pET43.1a + Seq1 + Seq2

We did 12 liquid cultures of 5 mL (6 of each).

08.11.2018

Miniprep pET43.1a + Seq1 + Seq2 and pBR322 + Seq8

We extracted our plasmids from the colonies set in liquid culture yesterday. We had:

• 5 colonies of pET43.1a + Seq1+ Seq2
• 6 colonies of pBR322 + Seq8
• 1 colony from negative control of pET43.1
• 1 colony from negative control of pBR322

(See here the Miniprep for plasmid extraction protocol) Results from NanoDrop (Blank used is buffer EB):
For pET 43.1a + Seq1 + Seq2

Colony	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	116.5	1.99
2	124.8	1.95
3	98.1	2.01
4	114.3	1.95
5	96.8	1.96

For pBR322 + Seq8

Colony	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	88.7	2.03
2	105.6	1.95
3	84.0	2.12
4	104.0	1.97
5	95.6	2.08
6	90.1	2.01

Colony	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
pET 43.1 negative control	110.6	1.98
pBR322 negative control	106.2	2.02

Observations: Concentrations are good enough for further use.

Digestion of pET43.1a + Seq1 + Seq2

Aim: Check that the ligation has successfully worked.
Restriction enzymes used: NdeI

Expected results:

pET43.1a	linearized	7.3 kB
pET43.1a + Seq1	1 fragment	6.63 kB
pET43.1a + Seq2	2 fragments	6.63 kB + 0.1 kB
pET43.1a + Seq1+ Seq2	2 fragments	6.63 kB + 1 kB

We digested 5 samples of our cloning products, and we also did some controls.

Procedure:

	Volume (μL)
DNA	5
Cut Smart	3
Ndel	1
H2O	21

Digestion of pBR322 + Seq8

Aim: Check that the ligation has successfully worked.
Restriction enzymes used: NdeI

Expected results:

pBR322	linearized	4 kB
pBR322 + Seq8	2 fragments	4 kB + 0.9 kB

We digested 6 samples of our cloning products, and 1 negative control.

Procedure:

	Volume (μL)
DNA	5
Cut Smart	3
Ndel	1
H2O	21

• Digestion at 37°C for 15 min
• Addition of 6 µL of 6X loading dye in each tube

Electrophoresis on agarose gel

We prepared an Agarose gel.
(See here the Agarose Gel Preparation protocol)

We filled:

• Wells 1 to 6: pBR322 + Seq8 (colonies 1 to 6)
• Well 7: pBR322 negative control
• Wells 8 to 12: pET43.1a + Seq1 + Seq2 (colonies 1 to 5)
• Well 13: pET 43.1 negative control

We let the DNA migrate for 10 minutes at 85 V and then 40 minutes at 125 V.

Results:

Remark: Date shift -2 days on photograph

Interpretations:

• For pBR322 + Seq8: On lanes corresponding to Col 1,3,4,5,6 two bands are clearly visible. One at 4 kb and another one at 1 kb, meaning that the plasmid digested by NdeI contained the insert Seq8. One band is visible on lane 2, Col 2 did not contain the insert. The control on lane 7 shows the plasmid digested without the insert at a higher molecular weight. The cloning worked for this construction, because bands observed between 700 bp and 1000 bp.
• For pET43.1a + Seq1 + Seq2: From lanes 8 to 11, only one band is visible, and bands are visible at the same molecular weight as the control one (pET43.1a w/o insert and digested with NdeI). On lane 12, the band is lightly lower than the others but no other band corresponding to an insert is visible on lane 12. The cloning did not work for this construction.

Liquid culture pET43.1a + Seq1 + Seq2 and pBR322 + Seq8

Colonies transformed with pET43.1a+Seq1+Seq2 (Colonies 7 to 25) were cultured in 5 mL LB media + 5 µL carbenicillin each (50 mg/mL)
Two culture of pBR322+ Seq8 (Col 3 and Col 4) were cultured in 25 mL LB media + 25 µl carbenicillin each (50 mg/mL)
(See here the Liquid Culture protocol)

Something was done one that day

08.06.2018

20 microchannel chips bonded to imaging dishes. 8 microchannel chips and 1 set of PDMS pieces for the well chip have been made.

Well chip fabrication protocol here .

Microchannel chip fabrication and bonding protocol here .

08.07.2018

20 bonded microchannel chips sterilized by UV exposure. 8 microchannel chips and 1 set of PDMS pieces for the well chip have been made. Preparation of vertical chip PDMS pieces.

Well chip fabrication protocol (the chip assembled on that day is slightly different but doesn't work) here .

Vertical chip fabrication protocol here .

Microchannel chip fabrication and sterilization protocol here .

08.09.2018

10 membrane microchannel chips have been made. Leaks made this a difficult operation. 8 microchannel chips and 1 set of PDMS pieces for the well chip have been made. Preparation of vertical chip PDMS pieces.

Membrane microchannel chip and basic microchannel chip fabrication protocol here .

Well chip fabrication protocol here .

Vertical chip fabrication protocol here .

08.10.2018

Membrane microchannel chips sterilized by UV exposure. Vertical chips bonded. 8 microchannel chips and 1 set of PDMS pieces for the well chip have been made. First successful vertical chip bonding, by modifying silver paste path.

Microchannel chip fabrication and sterilization protocol here .

08.13.2018

Miniprep pET 43.1a + Seq1 + Seq2
(See here the Miniprep for plasmid extraction protocol)
20 colonies from pET43.1a + Seq1 + Seq2 from petri dish (08.01.2018) were placed in liquid culture the day before (5 mL of LB + 10 µL of Carbenicillin).

Results from NanoDrop (Blank used is Buffer EB):

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	96.6	1.97
2	83.5	1.81
3	109.8	1.97
4	89.8	1.84
5	85.2	1.95
6	113.6	1.96
7	91.9	1.96
8	96.6	1.97
9	82.3	1.96
10	89.3	1.96
11	133	1.98
12	67.7	1.90
13	94.6	1.94
14	87.3	1.95
15	80.5	1.94
16	94.2	1.98
17	94.2	1.93
18	103	1.95
19	90.4	1.95
20	131.5	1.96

Digestion of pET 43.1a + Seq1 + Seq2

Reagents:

• pET 43.1a + Seq1 + Seq2
• Restriction enzymes : NdeI, PacI, BsmI, AvaI, NheI, BamHI, MscI, HindIII, XbaI, SpeI
• 10X Cut Smart
• Distilled water

1. Mix the following volumes of reagents and incubate at 37°C for 20 min

	Volume (μL)
pET 43.1a	42
10X Cut Smart	5
Distilled water	-
Ndel	1.5
Pacl	1.5

	Volume (μL)
pBR322	42
10X Cut Smart	5
Distilled water	-
Bsml	1.5
Aval	1.5

	Volume (μL)
Seq1	4.5
10X Cut Smart	5
Distilled water	31.5
Nhel	1.5
BamHI	1.5

	Volume (μL)
Seq2	8
10X Cut Smart	5
Distilled water	32
Mscl	1.5
HindIII	1.5

	Volume (μL)
Seq8	17.8
10X Cut Smart	5
Distilled water	24.2
Hhel	1.5
BamHI	1.5

2. Inactivate the digestion by heating the mixture in a water bath at 80 °C for 20 min

Electrophoresis on agarose gel

We prepared a 1 % agarose gel in order to separate the different fragments of the cut plasmids.
(See here the Agarose Gel Preparation protocol)

We filled:

• 2 wells with digested pET43.1a (n°1 and n°2)
• 10 wells with digested pET43.1a with NdeI (n°4 until n°13)

We let the DNA migrate for 10 min at 85 V on order to let the DNA penetrate the gel, and then 80 min at 125 V to efficiently separate the fragments.
We then revealed the gel under UV lamp.
Results of the agarose gel after migration:

Interpretations:

• pET43.1a has correctly been cut/linearized (lane 1 and 2). It was excised from the gel and extracted using Quiaquick Gel extraction kit.
• pET43.1a+ Seq1+seq2 cloning worked successfully on these colonies. We observed two bands as expected: one at ~ 6-7 kb and one at 1 kb.

We choose to do a Midiprep with sample 10 and 11 in order to have enough DNA for preparing samples for sequencing. We also transformed sample 11 in BL21(DE3) pLysS, our chassis.

Results from Nanodrop for pET43.1a

Concentration (ng/μL)	Abs(260 nm)/Abs(280 nm)
11.3	2.19
10.8	2.22

Midiprep pBR322+Seq8:

(See here the Miniprep for plasmid extraction protocol)

We extracted the plasmid from Col 3 and Col 4 from the cloning of pBR322+Seq8 (25 mL) from the 08.11.2018.
Results from Nanodrop:
For colony 3

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	41.0	1.86
2	2.6	1.90
3	38.3	1.91
4	-0.4	0.41
5	-1.1	0.67
6	1.0	1.44
7	-0.6	1.19
8	27.2	1.92

For colony 4

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	0.2	1.67
2	-0.6	0.99
3	52.5	1.92
4	-0.2	0.31
5	53.7	1.87
6	53.1	1.91
7	-0.6	1.67
8	-0.4	0.52

Sample 1,3,8 from Col 3 and sample 3,5,6 from Col 4 are kept for future sequencing. The other samples are thrown away. However, the concentrations are too low. Samples are put in liquid culture again for new DNA extraction.
The low concentrations can be due to a problem during the centrifugation steps, or to a too slow manipulation.

Bacterial transformation

(See here the Transformation of E. coli BL21 (DE3) pLysS protocol)
(See here the Transformation of E. coli DH5-alpha protocol)

3 transformations were processed:

• In BL21 (DE3)pLysS: pBR322 + Seq8 (Sample 11) and pET43.1a + Seq1 + Seq2 (Sample 3, Col4)
• In DH5α: Seq4

Bacterial stock preparation

(See here the Bacterial Stock protocol)
Launch of pre-cultures of DH5α strains (5 mL LB + carbenicillin (50 μg /mL) or 5 mL LB + chloramphenicol (25 μg/mL)).
Seq1, Seq2, Seq3, Seq5, Seq6, Seq7, Seq8, pET43, pBR322 and pVDL9.3

08.14.2018

Digestion of pBR322, Seq5, Seq6, Seq7

Reagents:

• pBR322, Seq5, Seq6, Seq7
• Restriction enzymes : XbaI, BamHI, MscI, SphI, HindIII, SpeI, BsmI, AvaI.
• 10X Cut Smart
• Distilled water

1. Mix the following volumes of reagents and incubate at 37°C for 20 min

	Volume (μL)
pBR322 (29.6 ng/μL)	42
10X Cut Smart	5
Distilled water	-
Bsml	1.5
Aval	1.5

	Volume (μL)
Seq5 (345 ng/μL)	12
10X Cut Smart	5
Distilled water	30
Nhel	1.5
BamHI	1.5

	Volume (μL)
Seq6 (1572 ng/μL)	6
10X Cut Smart	5
Distilled water	36
Mscl	1.5
HindIII	1.5

	Volume (μL)
Seq7 (1572 ng/μL)	36
10X Cut Smart	5
Distilled water	6
Hhel	1.5
BamHI	1.5

2. Inactivate the digestion by heating the mixture in a water bath at 80 °C for 20 min

Incubation for 20 min at 37°C

Inactivation for 20 min at 80°C

Addition of 6X loading dye

See the deposition order on the result picture

We let the gel run for 10 min at 85 V and then for 80 min at 125 V.
We revealed the gel with a UV light.

Results:

Observations: Digestion went well for all samples. We excised the interesting bands (inserts or plasmids) form the gel and did a gel extraction.
Results after excision:

Caption for fig 3:

• Wells 1-2: pBR322 cut
• Wells 4-5: Seq5 cut
• Wells 7-8: Seq6 cut
• Wells 10-11: Seq7 cut

(See here the Gel Extraction protocol)

Results from Nanodrop:

	[DNA] (ng/µL)	Abs(260 nm)/Abs(280 nm)
pBR322	11.4	2.49
Seq5	18.1	2.05
Seq6	11.9	3.10
Seq7	10.1	2.14

Bacterial stock preparation

(See here the Bacterial Stock protocol)

Culture of Seq1, Seq2, Seq3, Seq5, Seq6, Seq7, Seq8, pET43, pBR322 and pVDL9.3.
Measure the OD and when 0.6

Midiprep pBR322 + Seq8 and pET43.1 + Seq1 + Seq2

We extracted our plasmids from the colonies set in liquid culture yesterday. We had:

• 2 colonies of pET43.1a + Seq1 + Seq2
• 2 colonies of pBR322 + Seq8

(See here the Midiprep for plasmid extraction protocol)

Results from NanoDrop (Blank used is buffer EB):
pET 43.1a + Seq1 + Seq2 Col 10

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	7.4	2.11
2	58.5	1.91
3	0.8	3.57
4	3.2	1.26
5	64.5	1.90
6	2.8	1.72

pET 43.1a + Seq1 + Seq2 Col 11

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	1.2	3.17
2	0.2	-0.33
3	0.1	-0.2
4	1.0	1.63
5	0.7	3.02
6	79.9	-0.38

pBR322 + Seq8 Col 3

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	2.9	2.29
2	0	0.18
3	12.1	2.28
4	3.9	2.26
5	41.3	1.89
6	35.2	1.97

pBR322 + Seq8 Col 4

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	0.3	-0.84
2	33.8	1.93
3	33.2	1.88
4	34.1	1.93
5	0.8	4.15
6	27.8	1.92

Observations: Concentrations are not good.

• pET43.1a + Seq1 + Seq2, Col 10: Samples 2 and 5 have enough concentration.
• pBR322 + Seq8, Col 4: Samples 2,3,4,6 have enough concentration.

IN-FUSION ligation PBR322 + Seq5 + Seq6 + Seq7

We used the In-Fusion cloning kit provided by Ozyme in order to clone our three inserts Seq5 and Seq6 and Seq7 together in linearized pBR322. (see above).

1. Following the advice of the Takara Clontech technician we contacted, we first incubated the linearized vectors and inserts at 80°C for 5 min
2. We then placed it on ice for 3 minutes.
3. We mixed the following volumes into 1.5mL tubes.

Component	Cloning (μL)	Negative control (μL)	Positive control (μL)
90 ng Purified Seq5 (18.1ng/µl)	5	-	2 (control)
70 ng Purified Seq6 (14ng/µl)	5	-	2 (control)
70 ng Purified Seq7 (10.1ng/µl)	7	-	-
Linearized vector	10.5	1	1 (control)
5X In-Fusion enzyme premix	4	2	2
Deionized water	-	7	5

2. Incubate for 15 min in a water bath at 50°C
3. Ligation See here the Ligation protocol)
4. Set in ice
5. Transformation of the 3 plasmids into Stellar competent cells (provided with the cloning kit)
   • Competent cell volume : 33 μL
   • DNA volume: 2 μL
   (See here the transformation of E. coli DH5-alpha protocol)
6. Culture on petri dishes LB agar gel supplemented with carbenicillin overnight at 37°C

Liquid culture

(See here the Liquid Culture protocol)

We cultivated 2 colonies from the transformation of DH5α with commercial plasmid containing Seq4.

• One colony is cultivated in an Erlenmeyer in 25 mL LB medium + 25 μL carbenicillin (50 mg/mL)
• One colony is cultivated in two Falcon tubes, each containing 15 mL LB medium + 15 μL carbenicillin (50 mg/mL)

Since the results from this morning’s midiprep were not high enough, we cultivated again the results from cloning pBR322 + Seq8 and pET 43.1a + Seq1 + Seq2. We cultivated:

• Colony 3 of pBR322 + Seq8
• Colony 4 of pBR322 + Seq8
• Colony 11 of pET 43.1a + Seq1 + Seq2
• Colony 12 of pET 43.1a + Seq1 + Seq2

/p>

Each colony is cultivated in two Falcon tubes, each containing 15 mL LB medium + 15 μL carbenicillin (50 mg/mL), in order to do a midiprep.
The bacteria are cultivated overnight in a rotative incubator at 37°C, 180 rpm.

08.16.2018

Miniprep pBR322 + Seq5,6,7

We extracted our plasmids from the colonies set in liquid culture yesterday. We had 8 colonies of pBR322 + Seq5 + Seq6 + Seq7.
(See here the Miniprep for plasmid extraction protocol)

Results from NanoDrop (Blank used is buffer EB):

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	117.6	2.00
2	122.0	2.00
3	152.4	1.97
4	148.0	2.01
5	111.6	2.00
6	328.9	1.93
7	108	2.03
8	121.7	2.01

Observations: Concentrations are good, these samples can be saved for sequencing. (stored in the “SEQUENCING” box)

Digestion of pBR322 + Seq5,6,7

Aim: Check that the ligation has successfully worked.
Restriction enzymes used: BamHI

Expected results:

pBR322	linearized	4 kB
pBR322 + Seq5 + Seq6 + 7	2 fragments	4.8 kB + 2.3 kB

We digested 8 samples of our clonage products, and we also did some controls: pBR322 with BamHI, pBR322 w/o BamHI, Col 8 w/o BamHI.

	Volume (μL)
DNA	5
Cut Smart	3
BamHI	1
H2O	20

• Digestion at 37°C for 15 min
• Inactivation 80°C for 20 min.
• Addition of 6 µL of loading dye

Electrophoresis on agarose gel PBR322 + Seq5 + Seq6 + Seq7

We prepared a 1% agarose gel.
(See here the Agarose Gel Preparation protocol)

We deposited all the samples in the wells:

• Wells 1 to 8: pBR322 + Seq5,6,7 (colonies 1 to 8)
• Well 10: pBR322 + Seq5,6,7 Colony 8 w/o Enzyme
• Well 11: pBR322
• Well 12: pBR322 w/o enzyme

We let the gel run for 10 min at 85 V and 80 min at 125 V.
The gel was revealed with UV light.

Digested DNA from colonies migrated to the same molecular weight as the control one. No expected fragments were visible representing a digestion of our construction with BamHI.
None of the colonies contained the final construction. The cloning did not succeed.

We performed another ligation.

IN-FUSION ligation PBR322 + Seq5 + Seq6 + Seq7

We used the In-Fusion cloning kit provided by Ozyme in order to clone our three inserts Seq5 and Seq6 and Seq7 together in linearized pBR322. (see above).

1. We mixed the vector pBR322 and Seq5 and Seq7 and incubate at 80°C for 5 min. Then we add Seq7 and incubate 5 min at 70°C
2. We then placed it on ice for 3 minutes
4. We add the Enzyme Mix

Component	Cloning (μL)	Negative control (μL)	Positive control (μL)
90 ng Purified Seq5 (18.1ng/µl)	5	-	2 (control insert)
70 ng Purified Seq6 (14ng/µl)	5	-	2 (control)
70 ng Purified Seq7 (10.1ng/µl)	7	-	-
Linearized vector	10.5	1	1 (control vector)
5X In-Fusion enzyme premix	4	2	2
Deionized water	-	7	5

4. Incubate for 15 min in a water bath at 50°C
5. Ligation See here the Ligation protocol)
6. Set in ice
7. Transformation of the 3 plasmids into Stellar competent cells (provided with the cloning kit)
   • Competent cell volume : 33 μL
   • DNA volume: 2 μL
   (See here the transformation of E. coli DH5-alpha protocol)
8. Culture on petri dishes LB agar gel supplemented with carbenicillin overnight at 37°C

Digestion of pET43 + Seq3 + Seq4
/br> Aim: Obtain higher concentration of DNA after the gel extraction in order to perform efficiently the ozyme IN-FUSION kit.

1. Mix the following volumes of reagents and incubate at 37°C for 15 min

	Volume (μL)
pET43.1a	26
10X Cut Smart	5
Distilled water	16
Ndel	1.5
Pacl	1.5

	Volume (μL)
Seq3	29
10X Cut Smart	5
Distilled water	13
Nhel	1.5
BamHI	1.5

	Volume (μL)
Seq4	18
10X Cut Smart	5
Distilled water	24
Mscl	1.5
HindIII	1.5

2. Inactivate the digestion by heating the mixture in a water bath at 80 °C for 20 min.

Electrophoresis on agarose gel

We prepared a 0.8% agarose gel to separate the different fragments of the cut plasmids.
(See here the Agarose Gel Preparation protocol)

We filled:

• 2 wells with digested pET43.1a (n°3 and n°4)
• 2 wells with digested Seq3 (n°6 and n°7)
• 2 wells with digested Seq4 (n°9 and n°10)

We let the DNA migrate for 8 min at 85 V on order to let the DNA penetrate the gel, and then 65 min at 125 V to efficiently separate the fragments.
We then revealed the gel under UV lamp.

Interpretations:

• pET43.1a has not correctly been cut/linearized (lane 3 and 4). It was not excised from the gel and not extracted using Quiaquick Gel extraction kit.
• Seq3 has correctly been cut/linearized (lane 3 and 4). It was excised from the gel and extracted using Quiaquick Gel extraction kit.
• Seq4 has not correctly been cut/linearized (lane 3 and 4). It was not excised from the gel and not extracted using Quiaquick Gel extraction kit.
/


We choose to repeat the experiment for pET43, Seq3 and Seq4





08.17.2018





Miniprep pET 43.1a

(See here the Miniprep for plasmid extraction protocol) Results:

Colony	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	54.4	2.07
2	35.9	2.01
3	93.4	2.06
4	96.3	2.05
5	92.8	2.03
6	80.9	1.99
7	59.0	2.02
8	53.6	2.03
9	52.4	1.99




Transformation pBR322 + Seq5,6,7



Colony	Control +	Control -	Cloning
CFU	760	233	2




Liquid cultures

(See here the Liquid Culture protocol)

Culture of BL21(DE3)pLysS + pBR322+Seq8 (25 mL) + carbenicillin (50 μg/mL)
Culture of DH5alpha + pSB1C3 (25 ml) + chloramphenicol (25 μg/mL)
We also did some cultures on plate.

Culture of colonies 1 and 2 from ligation pBR322+Seq5,6,7 in 5 mL Falcon tubes





08.18.2018





Miniprep pBR322+Seq5,6,7
(See here the Miniprep for plasmid extraction protocol)

Results:

Colony	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	74	2.08
2	72.2	2.02





Digestion of pBR322 + Seq5,6,7

Aim: Check that the ligation has successfully worked.
Restriction enzymes used: BamHI, HindIII, SphI
Expected results:

	pBR322	linearized	4 kB
BamHI	pBR322 + Seq5 + Seq6 + Seq7	2 fragments	4.8 kB + 2.3 kB
pET43.1a + Seq1	pBR322 + Seq5 + Seq6 + Seq7	2 fragments	3.2 kB + 4.2 kB
pET43.1a + Seq1	pBR322 + Seq5 + Seq6 + Seq7	2 fragments	3.7 kB + 3.6 kB





We digested 1 samples of our cloning products, and we also did some controls pBR322 with BamHI.

Procedure:


	Volume (μL)
DNA	7
Cut Smart	3
Ndel	1
H2O	19







• Digestion at 37°C for 20 min
• Inactivation 80°C for 20 min
• Addition of 6 µL of 6X loading dye in each tube

Electrophoresis on agarose gel PBR322 + Seq5 + Seq6 + Seq7

We prepared a 1% agarose gel.
(See here the Agarose Gel Preparation protocol)
We let the gel run for 10 min at 85 V and 80 min at 125 V.











Digested DNA from colonies migrated to the same molecular weight as the control one. No expected fragments are visible representing a digestion of our construction with BamHI. None of the colonies contain the final construction. The cloning did not succeed.


Antibiotic test on liquid culture

Aim: Evaluate the effect of an overnight culture media containing carbenicillin on a non-resistant strain.

BL21- pBR322 + Seq8 were cultivated overnight on LB media containing carbenicillin. After centrifugation the supernatant was transferred into a new Erlenmeyer with 5 mL new LB media final volume 25 mL.
250 µL of overnight culture of DH5alpha – pSB1C3 were added to each media a OD was monitored.


	LB overnight culture	LB + CAM (chloramphenicol)	LB + CARB (carbenicillin)
Add DH5alpha-pSB1C3 liquid culture	250 µl	250 µl	250 µl
Expected growth	-	Yes	No




OD	LB overnight culture	LB + CAM	LB + CARB
t=0	0.092	0.049	0.039
t = 30 min	0.102	0.079	0.052
t = 1h	0.108	0.128	0.098
t = 1h30	0.115	0.242	0.142
t = 2h	0.124	0.390	0.127
t = 2h30	0.131	0.630	0.108
t = 3h	0.147	0.135	0.074





The antibiotic present in the overnight media did affect the growth of non-resistant bacteria.

Something was done one that day

08.13.2018

4 vertical chips bonded. Gold membrane PDMS well chips modification to enable current transmission.

Well chip fabrication protocol (now the well chips correspond with the protocol) here .

Vertical chip fabrication protocol here .

08.16.2018

Conductivity measurements of 2 gold-membrane PDMS well chips. Function generator was set on sine. The physical quantities measured here are Eg, the generator's tension amplitude and Ep, the voltage difference between the extremity of the platinum wire outside the well chip and a point on the edge of the membrane of the chip. the quantity calculated here is 20*log(Ep/Eg) for different frequencies.

Results

Interpretation

Voltage difference calculated is very low, indicating a very good conductivity for the gold-coated membrane. Technically, we measured the conductivity of the system membrane+platinum wire, but we showed that the wire's conductivity could be neglected. Resistance increases in higher frequencies, because of the skin-effect in metals: it transforms into an antenna. But as we are going to use only low frequencies, this doesn't affect us.

Well chip conductivity measurement protocol here .

08.20.2018

Liquid culture BL21 – RIP AND IPTG INDUCTION
Two colonies of transformed BL21 containing pBR322 and Seq8 (in order to produce RIP) are cultivated in 25 mL LB medium and 25 μL carbenicillin.
OD is followed regularly
Results:

OD	Col 3	Col 4
t=0	0.004	0.011
t = 30 min	0.010	0.015
t = 60 min	0.012	0.011
t = 160 min	0.019	0.029
t = 255 min	0.164	0.242
t = 300 min	0.426	0.562
t = 330 min	0.809	1.057
t = 355 min	1.184	1.369
	Dilution 30 mL LB	Dilution 30 mL LB
t = 405 min	1.285	1.491
t = 420 min	1.514	1.732

When the DO reaches 1.5 (and the bacteria are not in an exponential growth phase any more), we induced RIP production by adding IPTG.
(See here the IPTG Induction of protein expression protocol)

Different IPTG concentrations are tested for induction:

• 0.05 mM
• 0.1 mM
• 0.2 mM
• 0.5 mM

DO600 is measured every 30 minutes (the sample is diluted 1/10 before each measure)
Results:

Time after induction	0.05 mM	0.1 mM	0.2 mM	0.5 mM
t=0 min	1.51	1.51	1.73	1.73
t = 30 min	1.754	1.746	1.874	1.879
t = 60 min	2.118	2.219	2.346	2.265
t = 90 min	2.128	2.330	2.504	2.468
t = 120 min	2.552	2.025	2.630	2.493
t = 180 min	2.962	2.845	2.868	2.947

At t = 60 min et t = 120 min, 1 mL culture is taken, centrifuged and pellet and supernatant are conserved separately at -20°C, for further use.

Liquid culture of S. aureus
(See here the Liquid Culture protocol)

Liquid culture of S. aureus in 35 mL LB medium and 35 μL chloramphenicol.
We cultivated overnight in 37°C incubator.

Liquid culture

(See here the Liquid Culture protocol)

Liquid culture of DH5alpha- pSB1C3 and DH5alpha- pBR322 for plasmid stock.

08.21.2018

Digestion of pSB1C3 and sequences from EUROFINS GENOMICS (1 TO 8)

Reagents:

• Seq1 (conc. 493,7 ng/μL) : EcoRI and BamHI
• Seq2 (conc. 463,8 ng/μL): MscI and PstI
• Seq3 (conc. 179 ng/μL): EcoRI and BamHI
• Seq4 (conc. 354 ng/μL): MscI and PstI
• Seq5 (conc. 353 ng/μL): EcoRI and BamHI
• Seq6 (conc. 516 ng/μL): MscI and SphI
• Seq7 (conc. 305 ng/μL): HindIII and PstI
• Seq8 (conc. 325 ng/μL): EcoRI and PstI
• pSB1C3 from 2016 team (conc. 40 ng/μL) : XbaI and SpeI
• linearized iGEM plasmid pSB1C3 (conc. 25 ng/μL): XbaI and SpeI

Each digestion was performed in a total volume of 50 μL, containing 1500ng insert, 1.5 μL of each enzyme and 5 μL of CutSmart buffer.

Digestion was performed at 37°C for :

• 20 minutes for tubes without MscI
• 60 minutes for tubes containing MscI

Digestion was then inactivated at 80°C for 20 minutes.

Electrophoresis on agarOSE gel

A 0.8 % agar gel was prepared.
(See here the Agarose Gel Preparation protocol)

10 μL loading dye is added to each sample and each sample is put in two wells.

Results first gel:

Results after excision:

Results second gel: a second gel was run with digested Seq3, Seq4, Seq5, Seq6 and Seq7.
Migration for 10 minutes at 85V and then 1 hour at 125V.

Interpretations: all commercial plasmids have been successfully digested, except Seq4, for which the band corresponding to the insert we look for is unclear.

Gel extraction
(See here the Gel Extraction protocol)

Results from Nanodrop:

	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
Seq1 (cut)	31.6	2.00
Seq2 (cut)	23.9	1.92
Seq3 (cut)	16.8	2.00
Seq4 (cut)	3.3	3.11
Seq5 (cut)	32.1	1.98
Seq6 (cut)	25.8	1.91
Seq7 (cut)	24	1.97
Seq8 (cut)	10.8	2.25
pSB1C3 2016	7.6	2.7
pSB1C3 iGEM	9.0	2.07

IN FUSION kit for pSB1C3 + Seq1 +Seq2 and pSB1C3 + Seq8

(See here the Ligation protocol)
We used Ozyme cloning kit to insert Seq8 and Seq1 and 2 in iGEM plasmid.

Results: the volumes we mixed for the reaction

	ng	ng/μL	μL
pSB1C3	120	7.6	15
Seq1	85	31.6	3
Seq2	85	23.9	3.5
Enzyme mix	-	-	4

	ng	ng/μL	μL
pSB1C3	150	7.6	19
Seq8	70	15.7	5
Enzyme mix	-	-	4

Plasmids and inserts were mixed together and heated at 80°C for 5 min, and then set on ice for 3 min. Then the enzyme mix was added.
Samples were heated at 50°C for 15 min and then set on ice for 5 min.

Transformation in stellar competent cells

(See here the Transformation of E. coli DH5-alpha protocol)

Miniprep pbr322 + pSB1C3

Results from NanoDrop : Stock plasmid pSB1C3

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	161	1.95
2	143.9	1.95
3	129.1	1.95
4	265.3	1.92
5	290.7	1.97
6	156.3	1.96

Results from NanoDrop : Stock plasmid pBR322

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	139.4	1.98
2	221.0	1.92
3	139.8	1.94
4	138.4	1.99
5	129.1	1.95
6	120.1	1.95

Miniprep Cloning pBR322 + Seq5,6,7

Results from NanoDrop :

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	93.7	2.00
2	89.7	1.96
3	93.2	1.98
4	502.4	1.89
5	98.9	2.00
6	105.5	1.96
7	90.5	1.98
8	111.5	1.96
9	238	1.93
10	100.2	2.00

Digestion of pBR322 + Seq5,6,7

Aim: Check that the ligation has successfully worked on 10 other colonies.
Restriction enzymes used: BamHI

Expected results:

	pBR322	linearized	4 kB
BamHI	pBR322 + Seq5 + Seq6 + Seq7	2 fragments	4.8 kB + 2.3 kB

We digested 10 colonies of our clonage products, and we also did some controls: pBR322 with BamHI and without.

Procedure:

	Volume (μL)
DNA	7
Cut Smart	3
Ndel	1
H2O	19

• Digestion at 37°C for 20 min
• Addition of 6 µL of loading dye in each tube

Electrophoresis on agarose gel PBR322 + Seq5 + Seq6 + Seq7

We prepared a 1% agar gel. (See here the Agarose Gel Preparation protocol)
We let the gel run for 10 min at 85 V and 80min at 125 V.

Observations: Digested DNA from colonies migrated to the same molecular weight as the control one. No expecting fragments are visible representing a digestion of our construction with BamHI. None of the colonies contain the final construction. The cloning did not succeed.

08.22.2018

Results from cloning of pSB1C3 + Seq1 + Seq2 and pSB1C3 + Seq8

The two bacteria transformed with cloning results did grow on the agar plates.
(See here the Liquid Culture protocol)
10 colonies of each plate were placed in liquid culture and grew overnight.

Digestion pSB1C3 and pBR322

	Volume (μL)
pSB1C3	10.3
Cut Smart	5
H2O	28.7
Xbal	3
Spel	3

	Volume (μL)
pBR322	13.6
Cut Smart	5
H2O	25.4
Xbal	3
Spel	3

We let the samples digest for 30 minutes.

BL21 – NGF culture and protein induction

Yesterday’s preculture was centrifuged at 4500 x g for 15 minutes. Supernatant was discarded and pellet was washed with 5 mL fresh sterile LB medium. We centrifuged again for 10 minutes at 4500 x g and supernatant was discarded.
Pellet was resuspended in 10 mL LB medium + 10 μL carbenicillin and 10 μL chloramphenicol.

From this suspension, three liquid culture were launched in three 250 mL sterile Erlenmeyer.
Each Erlenmeyer contains:

• 35 mL LB medium
• 35 μL carbenicillin
• 35 μL chloramphenicol
• 1 mL resuspended pellet

The Erlenmeyers are incubated in a shaking incubator at 37 °C and DO is followed to induce protein expression when bacteria are in exponential growth phase.

At t = 80 min, when OD reaches 0.7, synthesis of the secretion system and NGF is induced by adding IPTG.
(See here the IPTG Induction of protein expression protocol)

Different IPTG concentrations are tested:

• Erlen 1 : 0.1 mM
• Erlen 2 : 0.5 mM
• Erlen 3 : 1 mM

The Erlenmeyer are incubated again, DO is measured every hour for two hours.
Two hours after induction, cultures are stopped and 1 mL of each culture is pipetted and put in a 1.5 mL Eppendorf tube.
Tubes are centrifuged, and pellet and supernatant are conserved separately.

Analysis of protein expression

We analyzed the protein present in the supernatant and in the cytoplasm of induced cells by SDS-Page gel.
(See here the SDS-PAGE protocol)

The wells were filled as following:

1. Culture medium before induction
2. Uninduced supernatant
3. Uninduced pellet
4. Culture medium after 0.1 mM induction
5. Supernatant after 0.1 mM induction
6. Pellet after 0.1 mM induction
7. Culture medium after 0.5 mM induction
8. Supernatant after 0.5 mM induction
9. Pellet after 0.5 mM induction

08.23.2018

Miniprep PSB1C3+ Seq8 and pSB1C3 + Seq1&2

Aim: extract plasmid from the colonies put in liquid culture yesterday (See here the Miniprep for plasmid extraction protocol)

Results from NanoDrop : plasmid pSB1C3 + Seq8

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	100.6	2.07
2	60	2.17
3	57	2.14
4	57.2	2.10
5	55	2.16
6	52.3	2.19
7	45.4	2.16
8	38.8	2.25
9	56.5	2.11
10	61.5	2.09

Results from NanoDrop : plasmid pSB1C3 + Seq1+2

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	40.6	1.99
2	49.6	1.99
3	71.5	1.95
4	63.7	1.99
5	55.6	2.03
6	46.8	1.98
7	86.6	1.96
8	74.9	1.93
9	57.1	1.97

Enzymatic digestion of the extracted plasmids

Aim: Verify that the cloning has correctly work for pSB1C3 + Seq8 and pSB1C3 + Seq1 + Seq2.
The following volumes have been mixed:
For pSB1C3 + Seq8:

	Volume (μL)
DNA	7
Cut Smart	3
H2O	18
EcoRI	1
PstI	1

For pSB1C3 + Seq1 + Seq2:

	Volume (μL)
DNA	7
Cut Smart	3
H2O	18
EcoRI	1
PstI	1

Gel electrophoresis of psb1c3 + Seq8

Digestion enzymes: EcoRI/PstI
(See here the Agarose Gel Preparation protocol)

Expected results:

pSB1C3	1 fragment	2070 bp
pSB1C3 + Seq8	2 fragments	2000 bp + 500 bp

Observations: Colony 2, 4, 5, 6, 8, 9, 10 acquired the plasmid and the insert.
BL21 cells were transformed with the plasmid form colony 2 and DH5α cells were transformed with plasmids from colony 2 and 4.
(See here the Transformation of E. coli DH5-alpha protocol)

Gel electrophoresis of pSB1C3 + Seq1,2

Digestion enzymes: EcoRI/PstI
(See here the Agarose Gel Preparation protocol)

Expected results:

pSB1C3	1 fragment	2070 bp
pSB1C3 + Seq8	2 fragments	2000 bp + 2260 bp

Observations: Colony 3, 7 and 8 did acquire the plasmid and the insert.

Antibiotic test on liquid culture

Aim: Evaluate the effect of an overnight culture media containing AMP on a non-resistant strain.

• Culture of BL21 + pBR322 + Seq8 (25 ml) + ampicillin (10 µg/mL)
• Culture of DH5alpha + pSB1C3 (5 ml) + chloramphenicol (50 µg/mL)
• Culture of DH5alpha + pSB1C3 (20 ml) + ampicillin (10 µg/mL)

Effect of AMP (10 µg/mL) on non-resistant cells:

• Overnight BL21 pBR322 + Seq8 AMP (10 µg/ml): OD = 5.78
• Overnight DH5a pSB1C3 AMP( 10 µg/ml): OD = 0.02

At 10 µg/ml Amp do not allow the growth of non-resistant bacteria.

After centrifugation the supernatant of Culture of BL21 + pBR322+Seq8 (25 ml) + AMP (50 µg/ml and 10 µg/ml) was transferred into a new erlenmeyer with 5ml new LB media final volume 25ml.
`
1/10 of overnight culture of DH5alpha – pSB1C3 were added to each media a OD was monitored.
Results:

	LB overnight culture AMP (50 µg/ml)	LB overnight culture AMP (10 µg/ml)	LB+ AMP (50 µg/ml) - 20 ml	LB+ AMP (50 µg/ml) - 10 ml	LB + CARB - 20 mL
Add DH5alpha-pSB1C3 liquid culture	250 µl	250 µl	200 µl	200 µl	200 µl
Expected growth	-	-	No	No	Yes

Results:

OD	LB overnight culture AMP (50 µg/ml)	LB overnight culture AMP (10 µg/ml)	LB+ AMP (50 µg/ml) - 20 ml	LB+ AMP (50 µg/ml) - 10 ml	LB + CARB - 20 mL
t=1h	0.116	0.094	0.082	0.091	0.087
t = 2h	0.2	0.172	0.043	0.231	0.258
t = 3h	0.272	0.231	0.034	0.317	0.839
t = 4h	0.342	0.307	0.023	0.137	0.839
t = 5h	0.369	0.349	0.021	0.092	1.33
t = 6h	0.426	0.394	0.014	0.079	1.54
t = 3h	0.467	0.477	0.017	0.063	1.71

The antibiotic present in the overnight media did affect the growth of non-resistant bacteria.

Liquid culture BL21 – NGF

Two colonies of BL21 transformed with our plasmid producing NGF were cultivated in 5 mL LB medium + 5 μL carbenicilline and 5 μL chloramphenicol.
(See here the Liquid Culture protocol)

08.24.2018

Miniprep PSB1C3+ Seq5,6,7 and Seq4

(See here the Miniprep for plasmid extraction protocol)

Results from NanoDrop: pSB1C3 + Seq5,6,7

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	352.9	1.93
2	308.4	1.93
3	336	1.93
4	325.1	1.93

Results from NanoDrop: pSB1C3 + Seq4:

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	95.8	2.05
2	104.8	1.99
3	87.9	2.01
4	101	1.95

Enzymatic digestion of PSB1C3 + Seq5,6,7

Aim: Verify that the IN-FUSION kit has correctly work for pSB1C3 + Seq5,6,7:

	Volume (μL)
DNA	7
Cut Smart	3
H2O	18
EcoRI	1
PstI	1

To verify that Seq6 is in the ligation:

	Volume (μL)
DNA	7
Cut Smart	3
H2O	18
Sphl	1
PstI	1

Digestion of Seq4:

	Volume (μL)
DNA (325 ng/µl)	30
Cut Smart	5
H2O	12
Mscl	1
HindIII	1

Gel electrophoresis of pSB1C3 + Seq5,6,7 and Seq4

Digestion enzymes: EcoRI/PstI and SphI/PstI
(See here the Agarose Gel Preparation protocol)

Expected results:

pSB1C3	1 fragment	2070 bp
EcoRI/PstI: pSB1C3 + Seq5,6,7	2 fragments	2070 bp + 3144 bp
EcoRI/PstI: pSB1C3 + Seq5,6,7	2 fragments	4500 bp + 762 bp
Seq4	2 fragments	2070 bp + 853 bp

Results :

Gel electrophoresis of pSB1C3 + Seq5,6,7 and Seq4

(See here the IPTG Induction of protein expression protocol)
From yesterday’s pre-culture, 800 mL culture of BL21 producing NGF was induced at 0.1 mM for NGF analysis.

S. aureus biofilm formation

S. aureus alone was plated in a 96-well plate in different concentrations in order to test different methods to wash the wells.
Plate is incubated at room temperature over the week end.

Something was done one that day

08.21.2018

Scanning electron microscopy (FEI Magellan 400 microscope) of a gold membrane.

Results

Interpretation

Membrane imaging confirmed the presence of 0.4 micrometer pores on the surface, enough to confine bacteria like E. coli.

08.22.2018

8 microchannel chips and 1 set of PDMS pieces for the well chip have been made.

Microchannel chip fabrication protocol here .

Well chip fabrication protocol here .

08.23.2018

PEDOT:PSS coated on alumina oxide membranes.

Results

Interpretation

The color of the alumina oxide membranes changed radically from light grey to black, suggesting the deposit of PEDOT:PSS on the membrane, as expected. Because of the lack of time and equipement, we won't try to verify that it is indeed PEDOT:PSS, however we are going to try to use electronic microscopy to visually evaluate the coating.

PEDOT:PSS coating protocol here .

08.24.2018

PEDOT:Ts and PEDOT:Cl coated on alumina oxide membranes.

Results

Interpretation

The color of the alumina oxide membranes changed radically from light grey to black with green shades for PEDOT:Cl and blue shades for PEDOT:Ts, suggesting the deposit of the polymers on the membranes, as expected. Because of the lack of time and equipement, we won't try to verify that it is indeed PEDOT:Ts and PEDOT:Cl, however we are going to try to use electronic microscopy to visually evaluate the coating.

PEDOT:Cl and PEDOT:Ts coating protocol here .

08.27.2018

Miniprep kill skitch
(See here the Miniprep for plasmid extraction protocol)

Results from NanoDrop: Kill Switch

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	661	1.86
2	889	1.87
3	665	1.87
4	1424	1.86
5	969	1.87
6	1093	1.87

Digestion of pSB1C3 + Seq5,6,7

Aim: Verify that the IN-FUSION kit has correctly work for pSB1C3 + Seq5,6,7:

	Volume (μL)
DNA	7
Cut Smart	3
Nhel	1
H2O	19

Electrophoresis on agarose gel of pSB1C3 + Seq5,6,7

Digestion enzymes: NheI
(Seehere the Agarose Gel Preparation protocol)

Interpretation: wells 1, 2 and 4 have the expected fragments. The cloning did work for the three inserts.

Transformation

(See here the Transformation of E. coli DH5-alpha protocol)
BL21(DE3) pLysS cells were transformed with the plasmid (pSB1C3 + Seq 5,6,7) from colony 2 and DH5α cells were transformed with plasmids (pSB1C3 + Seq 5,6,7 and pSB1C3 + Seq 1,2) from colony 2 and 7.

Digestion Ks and pSB1C3

	Volume (μL)
Ks (665ng/ μL)	9.3
Cut Smart	5
EcoRI	1.5
PstI	1.5
H2O	42

	Volume (μL)
pSB1C3 (156ng/ μL)	16
Cut Smart	5
Xbal	1.5
Spel	1.5
H2O	26

Liquid culture

(See here the Liquid Culture protocol)
DH5alpha + pSB1C3 + Seq8

Biofilm formation test

Two colonies of BL21(DE3) pLysS cells that were transformed with pSB1C3+Seq8 were put in liquid culture in 30 mL LB medium with 30 μL chloramphenicol (25 mg/mL).
Two colonies of S. aureus were also put in liquid culture in 30 mL LB medium with 30 μL chloramphenicol (25 mg/mL).

Results (DO is followed during the culture):

	BL21 + pSB1C3 RIP	S. aureus
t=0	0	0
t = 90 min	0	0
t = 180 min	0.002	0.008
t = 240 min	0.002	0.023
t = 300 min	0.025	0.076
t = 360 min	0.120	0.281
t = 430 min	0.416	0.731
t = 550 min	cf. below	1.873

When BL21(DE3) pLysS cells are in exponential growth phase (OD600 nm =0.416), the culture is separated into four 5 mL cultures in 50 mL Erlenmeyer. RIP production is induced with different concentrations of IPTG:

• One is not induced (negative control)
• One is induced with 0.1 mM
• One is induced with 0.5 mM
• One is induced with 1 mM

After two hours of induction (i.e. 550 minutes in the protocol above), OD600 nm is measured again:

	uninduced	IPTG 0.1 mM	IPTG 0.5 mM	IPTG 1 mM
OD600 nm/td>	2.495	2.464	2.417	2.337

1 mL of each culture is kept intact in a 1.5 mL Eppendorf tube and put at 4°C waiting for further treatment.
The 4 mL left are centrifuged, supernatant is collected.

S. aureus is then grown overnight in a 96-well plate in different conditions in order to test the effect of both supernatant and our bacteria.
Each well contains 100 μL of the following mix:

1. A-B LB medium alone

1. E-H 990 μL LB + Cm + 10 μL S. aureus culture
2. 990 μL LB un-induced supernatant + 10 μL S. aureus culture
3. 990 μL LB 0.1 mM-induced supernatant + 10 μL S. aureus culture
4. 990 μL LB 0.5 mM-induced supernatant + 10 μL S. aureus culture
5. 990 μL LB 1 mM-induced supernatant + 10 μL S. aureus culture
6. 890 μL LB + Cm + 100 μL uninduced BL21 + 10 μL S. aureus culture
7. 890 μL LB + Cm + 100 μL 0.1 mM induced BL21 + 10 μL S. aureus culture
8. 890 μL LB + Cm + 100 μL 0.5 mM induced BL21 + 10 μL S. aureus culture
9. 890 μL LB + Cm + 100 μL 1 mM induced BL21(DE3)pLysS + 10 μL S. aureuss culture

800 mL culture NGF purification on a Nickel-NTA column

(See here the Purification of his-tagged protein from culture medium protocol)
After purification, 10 fractions of 1 mL were collected from the column elution.

SDS-PAGE of the collected fractions & Western blot for NGF production analysis

This SDS-PAGE didn’t give satisfying results. There was a misunderstanding of the purification protocol as we had let the charging of the column go on over the weekend, which led to the emptying of the nickel from the column.

08.28.2018

Miniprep DH5alpha pSB1C3 Seq8 colony 2 and 4
(See here the Miniprep for plasmid extraction protocol)

Results from NanoDrop: DH5alpha pSB1C3 Seq8 colony 2

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	210.7	1.97
2	234.9	1.94
3	201.2	1.94
4	210.7	1.95
5	217.1	1.93

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	222.7	1.95
2	264.4	1.95
3	224.2	1.95
4	212.4	1.96
5	225.3	1.94

Store at -20°C in the box named "séquençage".

Liquid culture

(See here the Liquid Culture protocol)

• 1 colony of DH5alpha + pSB1C3 + Seq1,2 (colony 7)
• 1 colony of DH5alpha + pSB1C3 + Seq5,6,7 (colony2)
• 2 colonies of BL21 + pSB1C3 + Seq5,6,7 (colony 2)
• 1 colonies S. aureus

Electrophoresis on agarose gel

Digestion enzymes for pSB1C3: XbaI + SpeI
Digestion enzymes for Ks: EcoRI + PstI

(See here the Agarose Gel Preparation protocol)
Results:

Interpretation: three bands are visible for pSB1C3, we will not excise this one. We excised the lower band for the kill switch

Gel extraction

(See here the Gel Extraction protocol)

Results from Nanodrop:

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
Ks cut	8.4	2.66

IN FUSION kit for pSB1C3 + Ks

(See here the Ligation protocol>

Sample	mass (ng)	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
pSB1C3	140	6.9	20
Ks	60	8.4	7
Enzyme mix	-	-	4

Transformation in STELLAR competent cells

(See here the Transformation of E. coli DH5-alpha protocol)

Midiprep S. aureus

(See here the Midiprep for plasmid extraction protocol)
Results from NanoDrop: S. aureus

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	222.7	1.95
2	264.4	1.95
3	224.2	1.95
4	212.4	1.96

Purification of NGF from the bacterial pellet of an 800 mL culture using sonication and FPLC

(See here the Fast Protein Liquid Chromatography (FPLC) protocol)

Tomorrow will be deposited on an SDS-PAGE gel the fractions:

• A2 (flow through from the deposited proteins)
• B3 (flow through from the elution at a 5% buffer B proportion)
• A4, A5, A6, A7 (2 mL fractions around the A280 peak)

Kill switch test #1

Cells pre-culture of DH5a + Seq9 + Carbenicillin (25 µg/mL) at different temperatures.
Three culture conditions were performed: 37°C, 15°C and 4°C without positive control. After 4 hours of incubation 5 ml of each samples were placed at 37°C, 15°C or 4°C and incubated overnight.

Bacterial cultures placed at 15°C did not grow up on the four-hour period compared to the one a 37°C. Once placed at 37°C all bacterial cultures grown up again even if it is only slightly for the ones placed at 15°C and 4°C.
We supposed the Kill switch has a bacteriostatic effect. We will perform another test with a positive control and different temperatures.

08.29.2018

Miniprep DH5alpha pSB1C3 Seq1,2 colony 7 and DH5alpha pSB1C3 Seq5,6,7 colony 2

(See here the Miniprep for plasmid extraction protocol)
Results from NanoDrop: DH5alpha pSB1C3 Seq1,2 colony 7

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	346.1	1.92
2	391.7	1.91
3	411.2	1.92
4	330.8	1.92
5	321.9	1.90

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	272	1.95
2	242.3	1.93
3	261.7	1.94
4	283	1.92
5	279.8	1.93

Store at -20°C in the box named "séquençage".
Results from NanoDrop : Kill Switch

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	9.9	2.01
2	8.1	2.12
3	6.7	2.7
4	10.1	2.14
5	9.7	1.90

Store at -20°C in the box named S. aureus

Digestion of DH5alpha pSB1C3 + Seq5,6,7

Aim: Verify that the In-Fusion kit has correctly worked for DH5alpha pSB1C3 + Seq5,6,7

	Volume (μL)
DNA	7
Cut Smart	3
Nhel	1
H2O	19

Digestion of DH5alpha pSB1C3 + Seq5,6,7

Aim: Verify that the In-Fusion kit has correctly worked for DH5alpha pSB1C3 + Seq5,6,7

	Volume (μL)
DNA	7
Cut Smart	3
EcoRI	1
PstI	1
H2O	18

Electrophoresis on agarose gel of DH5alpha pSB1C3 + Seq1,2 and DH5alpha pSB1C3 + Seq5,6,7

(See here the Agarose Gel Preparation protocol)

Results:

BL21(DE3)pLysS liquid culture and RIP production induction

Two colonies of BL21(DE3)PlysS cells transformed with pSB1C3+Seq8 were put in liquid culture in 25 mL LB medium with 25 μL chloramphenicol (25 mg/mL).

Results: OD is followed during the culture

	BL21(DE3)pLysS + pSB1C3 RIP
t = 0 min	0.248
t = 90 min	1.550

Culture is diluted 1/10, making the OD600nm 0.228.
When OD reaches 0.667, BL21(DE3)pLysS culture is separated into four 5 mL cultures in 50 mL Erlenmeyer. RIP production is induced with different concentrations of IPTG:

• One is not induced (Negative control)
• One is induced with 0.1 mM
• One is induced with 0.5 mM
• One is induced with 1 mM

Results after two hours induction (i.e. 550 minutes in the protocol above), OD is measured again:

	uninduced	IPTG 0.1 mM	IPTG 0.5 mM	IPTG 1 mM
OD600 nm/td>	2.011	1.830	1.741	1.868

1 mL of each culture is kept intact in a 1.5 mL Eppendorf tube and put at 4°C waiting for further treatment.
The 4 mL left are centrifuged, supernatant is collected.

SDS-PAGE of the protein fractions from the FPLC purification to prepare the Mass Spectrometry samples

(See here the SDS-PAGE protocol)

Deposit plan and results:

To analyze this gel, we will perform a Western Blot tomorrow.

08.30.2018

Miniprep STELLAR pSB1C3 Seq5,6,7 colony 1,2,3,4 and STELLAR pSB1C3 Seq9 (Ks)

(See here the Miniprep for plasmid extraction protocol)

Results from NanoDrop: STELLAR pSB1C3 Seq5,6,7 colony 1,2,3,4

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	93.2	2.01
2	112	1.95
3	114	1.94
4	109	2.01

Results from NanoDrop: STELLAR pSB1C3 Seq9

Sample	DNA Concentration (ng/µL)	Abs(260 nm)/Abs(280 nm)
1	83.4	2.01
2	156.5	1.88
3	69.2	1.98
4	103	1.98
5	98.3	2.00
4	123.7	1.97

Digestion of STELLAR pSB1C3 + Seq5,6,7

Aim: Verify that the In fusion kit has correctly work for STELLAR pSB1C3 + Seq5,6,7

	Volume (μL)
DNA	7
Cut Smart	3
Nhel	1
H2O	19

Aim: Verify that the In fusion kit has correctly work for STELLAR pSB1C3 + Seq9

	Volume (μL)
DNA	7
Cut Smart	3
PstI	1
EcoRI	1
H2O	19

Electrophoresis on agarose gel of STELLAR pSB1C3 + Seq5,6,7 and STELLAR pSB1C3 + Seq9

(See here the Agarose Gel Preparation protocol)
Results:

Biofilm formation test

S. aureus is now grown overnight in a 96-well plate in different conditions in order to test the effect of both supernatant and our bacteria.
Each well contains 100 μL of the following mix (using the pellet suspension and supernatant from yesterday’s culture):

1. A-B LB medium alone and E-H 990 μL LB + Cm + 10 μL S. aureus culture
2. A-D 990 μL LB + Cm + 10 μL S. aureus culture + 0.2 μL IPTG (0.5 M) and E-H 990 μL LB + Cm + 10 μL S. aureus culture + 1 μL IPTG (0.5 M)
3. A-D 990 μL LB + Cm + 10 μL S. aureus culture + 2 μL IPTG (0.5 M)
4. A-D LB + BL21 without plasmid + S. aureus
5. E-H LB + BL21 without plasmid + S. aureus + IPTG (1 mM)
6. 990 μL LB un-induced supernatant BL21-RIP + 10 μL S. aureus culture
7. 990 μL LB 0.1 mM-induced supernatant BL21-RIP + 10 μL S. aureus culture
8. 990 μL LB 0.5 mM-induced supernatant BL21-RIP + 10 μL S. aureus us culture
9. 990 μL LB 1 mM-induced supernatant BL21-RIP + 10 μL S. aureus culture
10. 890 μL LB + Cm + 100 μL uninduced BL21 + 10 μL S. aureus culture
11. 881 μL LB + Cm + 109 μL 0.1 mM induced BL21 + 10 μL S. aureus culture
12. 875 μL LB + Cm + 115 μL 0.5 mM induced BL21 + 10 μL S. aureus culture
13. 880 μL LB + Cm + 110 μL 1 mM induced BL21 + 10 μL S. aureus culture

To identical plates are incubated at 37°C overnight.

Transfer and immunodetection on membrane of the SDS-PAGE Gel from the 08.29.18

(See here the Gel Transfer for Western Blotting protocol)

Results:

At around 33 kDa, one band more intense seems to be the NGF.

The result of this Western Blot is not very clear. Yet, there seems to be a band at 33 kDa in moleculrar weight (MW) (which is the size of un-cleaved proNGF with the signal sequence HLYA). Several other bands of upper and lower MW bands are also visible. To confirm that the expression of recombinant proNGF is effective, this band along with a set of lower MW ones will be studied by Mass Spectrometry as shown below. If confirmed, this will mean that our NGF is not retained on the Ni-NTA column for an unknown reason, and that our proNGF is not cleaved from the signal peptide by the TEV protease. Presence of proNGF in lower bands might mean that the signal is cleaved, or that the full length protein has suffered from protease attack.

08.31.2018 • Test Kill Switch #2 1. From overnight pre-culture of Stellar pSB1C3+ KS and pSB1C3 backbone (positive control) in LB+ Chloramphenicol 25 μg/mL (CAM)

	Stellar pSB1C3+KS	Stellar pSB1C3
OD(600 nm)	1.240	1.660

Launch of 8 bacterial cultures in Falcon 50 of 15 ml + CAM, with pre-cultures diluted 1/30.
Incubation all weekend at 180 rpm at different temperatures: 37°C, 25°C, 20°C and 18°C.

Test biofilm formation on various metals #1

In order to test the adherence of our biofilm to metallic surfaces, two cylindrical pieces of metal, one made of titanium (TA6V4) and the other of stainless steel (316L), approximately 5 cm in length and 1.0 cm in diameter each (Generous gift from I-CERAM, Limoges, France), were incubated in Falcon 15 + 7 ml of LB+ CAM. 50 µl of BL21(DE3)pLysSeq5,6,7 were inoculated. Incubation over the weekend at 37°C, 180 rpm.

Biofilm in 96-wells plate

< One of the two plates that was incubated yesterday was washed with the following procedure:

• Supernatant in each well was discarded with a pipet
• Each well was then washed two times by adding 100 μL of distilled water

Plates were read by measuring fluorescence at 510 nm before and after washing. We obtained the following results:
The washed plate was filled again with 100 μL LB + CAM in each well, and left to incubate for the week-end at 37°C. The second plate was left for the week-end as well.