Team:Pasteur Paris/Notebook

""

Week 28 9 - 15 July
Week 29 16 - 22 July
Week 30 23 - 29 July
Week 31 30 July - 5 Aug
Week 32 6 - 12 Aug
Week 33 13 - 19 Aug
Week 34 20 - 26 Aug
Week 35 27 Aug - 2 Sept
Week 36 3 - 9 Sept
Week 37 10 - 16 Sept
Week 38 17 - 23 Sept
Week 39 24 - 30 Sept
Week 40 1 - 7 Oct
Week 41 8 - 14 Oct
Week 42 14 - 17 Oct

Microfluidics/Membrane



07.10.2018


Institut Curie’s lab MMBM at IPGG (Institut Pierre Gilles de Gennes) hands us the molds for the microchannel chip and grants 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.


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.


07.12.2018


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


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.

Figure 1: Strips before cutting
Figure 1: Strips before cutting
Figure 2: Cut strip with scratches
Figure 2: Cut strip with scratches

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.


Figure 3: Microchannel chip bonded to imaging dish
Figure 3: Microchannel chip bonded to imaging dish

Bacteriology

Cell culture

Microfluidics/Membrane



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
Figure 1: Seq #8 – T7_RIP
Figure 1: Seq #8 – T7_RIP
Figure 2: Seq#1 – 3a_NGF_Part1
Figure 2: Seq#1 – 3a_NGF_Part1
Figure 3: Seq#2 – 3a_NGF_Part2
Figure 3: Seq#2 – 3a_NGF_Part2

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.


19.07.2018


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


Bacteriology

Cell culture

Microfluidics/Membrane


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

Figure 1: Picture of the 3 petri dishes at t = 0.
Figure 1: Picture of the 3 petri dishes at t = 0.


  1. 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
Figure 2: Seq5 – RIP_Part1
Figure 2: Seq #8 – T7_RIP
Figure 3: Seq7 – RIP_Part3
Figure 3: Seq7 – RIP_Part3
Figure 4: pBR322
Figure 4: pBR322

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
Figure 5: Picture of the 3 petri dishes at t = 24.
Figure 5: Picture of the 3 petri dishes at t = 24.


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:

Figure 6: Picture of the petri dish (with 50 μL of culture) 24 hours after transfer on another filter.
Figure 6: Picture of the petri dish (with 50 μL of culture) 24 hours after transfer on another filter. .

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.


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

Figure 1: Conductivity of a platinum wire for different frequencies
Figure 1: Conductivity of a platinum wire for different frequencies

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 used in PDMS well chips. Resistance increases in higher frequencies, because


07.27.2018


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


Bacteriology

Cell culture

Microfluidics/Membrane


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


  1. 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:

Figure 1:
Figure 1:

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.


Figure 1-2: gel after DNA extraction of pBR322 and of Seq8
Figure 1-2: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


  1. Incubate for 15 min in a water bath at 50°C
  2. Ligation See here the Ligation protocol)
  3. Set in ice
  4. 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)
  5. 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

Figure 2: Negative Control
Figure 2: Negative Control
Figure 3: Positive Control
Figure 3: Positive Control
Figure 4: Ligation of pBR322 and Seq8 (T7-RIP)
Figure 4: Ligation of pBR322 and Seq8 (T7-RIP)


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:

    Figure 5:
    Figure 5:
  • 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:

Figure 6:

/br>


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


  1. 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.

Figure 7: Electrophoresis gel 0.8% (1. 2. pET43.1a / 3. 4. Seq1 / 5. 6. Seq2) before DNA extraction
Figure 7: Electrophoresis gel 0.8% (1. 2. pET43.1a / 3. 4. Seq1 / 5. 6. Seq2) before DNA extraction

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:

Figure 8: 0.8% Agarose gel after excision of the DNA
Figure 8: 0.8% Agarose gel after excision of the DNA

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

  1. Incubate for 15 min in a water bath at 50°C
  2. Ligation (See Ligation protocol)
  3. Set on ice
  4. 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)

  5. 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


  1. 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.

Figure 9: Electrophoresis gel 0.8 % before DNA extraction of pBR322, Seq5, Seq6, Seq7
Figure 9: ElectroElectrophoresis gel 0.8 % before DNA extraction of pBR322, Seq5, Seq6, Seq7

  • 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:

    Figure 10: Agarose gel electrophoresis after excision of the DNA
    Figure 10: Agarose gel electrophoresis after excision of the DNA


    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


    1. Incubate for 15 min in a water bath at 50°C
    2. Ligation See here the Ligation protocol)
    3. Set in ice
    4. 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)
    5. 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.

    Figure 11: Petri dish with the 6 colonies that were placed in liquid culture
    Figure 11: Petri dish with the 6 colonies that were placed in liquid culture






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.


07.31.2018


2 additional PDMS wells assembled. 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.


08.01.2018


Conductivity tests on membranes assembled on chips. Well chips not functional. 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


Bacteriology

Cell culture

Microfluidics/Membrane

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:

Figure 1: Agarose gel


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:

Figure 2: Agarose gel


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


  1. Incubate for 15 minutes in a water bath at 50°C
  2. Ligation (See Ligation protocol)
  3. Set in ice
  4. Transformation of the 3 plasmids into Stellar competent cells (provided with the clonage kit):
    • Competent cell volume: 50 μL
    • DNA volume: 2 μL
  5. 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:

Figure 3: Agarose gel.
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



  1. 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:

Figure 4:  Agarose gel 1% after electrophoresis
Figure 4: Agarose 1% gel after electrophoresis


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)

Figure 5: Agarose gel 1% after electrophoresis of digested pBR322, pET43.1a, seq#1, seq#2 and seq#8 and extraction of the sequences of interest.
Figure 5: Agarose gel 1% after electrophoresis of digested pBR322, pET43.1a, seq#1, seq#2 and seq#8 and extraction of the sequences of interest.
(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


  1. Incubate for 15 minutes in a water bath at 50°C.
  2. Set in ice.
  3. Ligation (See here the Ligation protocol)
  4. 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)
  5. 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

Figure 6: pBR322 + NegControl and pBR322 Seq8.
Figure 6: pBR322_ NegControl and pBR322 Seq8.
Figure 7: pET43.1 + Seq1+2 and pET43.1_ NegControl.
Figure 7: pET43.1 + Seq1+2 and pET43.1_ NegControl.
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:

Figure 8:


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.


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.


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.


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.


Bacteriology

Cell culture

Microfluidics/Membrane



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



  1. 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:

Figure 1:  Agarose gel 1% after electrophoresis of digested pBR322, pET43.1a, seq#1, seq#2 and seq#8.
Figure 4: Agarose gel 1% after electrophoresis of digested pBR322, pET43.1a, seq#1, seq#2 and seq#8.


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



  1. 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:

    Figure 2: Agarose 0.8% gel after electrophoresis of digested pET43.1+Seq1+Seq2
    Figure 2: Agarose 0.8% gel after electrophoresis of digested pET43.1+Seq1+Seq2


    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:

    Figure 3: Agarose 0.8% gel after electrophoresis of digested pET43.1+Seq1+Seq2 after excision
    Figure 2: Agarose 0.8% gel after electrophoresis of digested pET43.1+Seq1+Seq2 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


    1. Incubate for 15 min in a water bath at 50°C
    2. Ligation See here the Ligation protocol)
    3. Set in ice
    4. 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)
    5. 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.

    Figure 4: Agarose gel


    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
    3. 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


    1. Incubate for 15 min in a water bath at 50°C
    2. Ligation See here the Ligation protocol)
    3. Set in ice
    4. 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)
    5. 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


    1. 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.

    Figure 5: Agarose gel

    Figure 4: Agarose gel


    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
        8 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.


        Figure 7: Agarose gel


        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.


    08.16.2018


    Conductivity measurements of the 3 gold-membrane PDMS well chips. Success.


    Bacteriology

    Cell culture

    Microfluidics/Membrane




    Something was done one that day





    Something was done one that day




    08.22.2018


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


    08.23.2018


    PEDOT:PSS coated on alumina oxide membranes. Success.


    08.24.2018


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


    Bacteriology

    Cell culture

    Microfluidics/Membrane




    Something was done one that day





    Something was done one that day




    08.27.2018


    4 well chips have been made, each with a different type of membrane alumina oxide membrane filter (bare, PEDOT:PSS, PEDOT:Cl, PEDOT:Ts). 4 vertical chips bonded to tissue culture dishes.


    08.28.2018


    8 double membrane microchannel chips have been bonded to imaging dishes and sterilized by exposure to UV rays. Leaks made this a difficult and time-consuming operation. 8 microchannel chips have been made.


    Bacteriology

    Cell culture




    Something was done one that day





    Something was done one that day





    Something was done one that day


    Bacteriology

    Cell culture

    Microfluidics/Membrane




    Something was done one that day





    Something was done one that day




    09.11.2018


    Bonding to imaging dishes and UV sterilization of 20 microchannel chips.


    Bacteriology

    Cell culture




    Something was done one that day





    Something was done one that day


    Bacteriology

    Cell culture




    Something was done one that day





    Something was done one that day


    Bacteriology

    Cell culture

    Microfluidics/Membrane




    Something was done one that day





    Something was done one that day




    10.04.2018


    Liquid culture of BL21. Biofilm culture on 6 PDMS well chips (membranes: 2 gold, 1 alumina oxide, 1 PEDOT:PSS, 1 PEDOT:Cl and 1 PEDOT:Ts).


    10.05.2018


    Biofilm growth measurement. Liquid culture of BL21 and biofilm culture on 6 PDMS well chips.

    Membrane microchannel chip bacteria confinement experiment. Optical density measure of a sample of liquid culture. Liquid culture was poured in a microchannel chip on one side, and optical density of the liquid that flowed to the other side of the microchannnels was measured.

    Results

    Liquid culture OD: 0.44

    OD of liquid after flowing through the chip: 0.41.

    Interpretation

    We expected a much lower OD after liquid flow through the chip, so this suggests the presence of a leak in the chip, that allows the liquid culture to flow without retaining the bacteria. The decision was taken to investigate further and to observe the flow under a microscope.


    Bacteriology

    Cell culture

    Microfluidics/Membrane




    Something was done one that day





    Something was done one that day




    10.08.2018


    Liquid culture of BL21. Biofilm culture on 6 PDMS well chips

    Biofilm growth measurement.


    10.11.2018


    Liquid culture of BL21. Biofilm culture on 6 PDMS well chips

    Biofilm growth measurement.


    10.12.2018


    Liquid culture of BL21. Biofilm culture on 6 PDMS well chips

    Biofilm growth measurement.

    Measurement of the electrical resistance of the membranes with the biofilm.


    Bacteriology

    Cell culture

    Microfluidics/Membrane




    Something was done one that day





    Something was done one that day




    10.15.2018


    Liquid culture of BL21. Biofilm culture on 6 PDMS well chips

    Biofilm growth measurement.

    Measurement of the electrical resistance of the membranes with the biofilm.

    Measurement of the membrane's electrical resistance without biofilm.

    Results: membrane conductivity

    Function generator was set on square at 200 Hz. The physical quantities measured are Eg, the generator tension amplitude, and Ep, the amplitude of the voltage difference between a point on the membrane inside the well and the extremity of the platinium strip outside the well. Tension amplitude of the resistor is given by Er = Eg - Ep. Current flowing through the electric circuit is calculated with I = Er/R. Conductivity of the membrane is given by I/Ep. Conductivity of each membrane was measured 3 times. Figure 1 shows the mean value for each membrane and the standard deviation.


    Figure 1: Membrane conductivity
    Figure 1: Membrane conductivity

    Interpretation

    Bare alumina oxide and PEDOT:PSS-coated membranes show similar conductivies, indicating the incomplete coating of PEDOT:PSS on alumina oxide membranes. On the opposite, PEDOT:Cl and PEDOT:Ts seem to exhibit on average better conductivities, but in the same time, the coating of these membranes revealed by electron microscopy seemed to have covered the alumina oxide membranes in a more uniform way, assuring enhanced conductive capabilities. These results can be criticized because of the high deviation and because the membranes conductivity was measured after several biofilms were grown on them, which may have affected the measurements.


    10.16.2018


    Measurement of the electrical resistance of the membranes with the biofilm. Measurement of the biofilm's growth.


    Results: biofilm growth

    Biofilm growth was measured 4 times in total. For each series of measure, the measured optical densities were divided by the optical density of the base liquid culture, to normalize the measures. Figure 2 shows the mean value for each type of membrane and the standard deviation.

    Figure 2: Biofilm growth
    Figure 2: Biofilm growth

    Results: biofilm conductivity

    For conductivity measurements, function generator was set on square at 200 Hz. The physical quantities measured are Eg, the generator tension amplitude, and Ep, the amplitude of the voltage difference between a point on the biofilm inside the well and the extremity of the platinium strip outside the well. Tension amplitude of the resistor is given by Er = Eg - Ep. Current flowing through the electric circuit is calculated with I = Er/R. Conductivity of the membrane is given by I/Ep. Conductivity of each membrane with a biofilm was repeated 3 times. Figure 4 shows the average conductivity of the membrane with the biofilm, and the standard deviation.

    Figure 3: Membrane conductivity with biofilm
    Figure 3: Membrane conductivity with biofilm

    To approximate very roughly the conductivity of the biofilm, the average conductivity values of the membranes with a biofilm were divided by the corresponding average biofilm growth values, and the conductivity of the membrane was then substracted. Figure 4 shows the estimated biofilm conductivity.


    Figure 4: Biofilm conductivity
    Figure 4: Biofilm conductivity

    Interpretation

    As told by the membrane manufacturer, biofilm formation on gold membranes seems indeed to be more difficult than on other membranes. However we expected PEDOT:PSS-coated membranes to stimulate more the growth of biofilm, but perhaps that may be just another indicator of the imcomplete coating. Surprisingly, PEDOT:Cl tends to allow better formation of biofilms. We realized only after the experiments the need for a control biofilm culture without membrane.

    Conductivity with a biofilm is better with gold membranes, although the conductivity of gold membranes themselves isn't the best. This may be explained by the fact, that because of the thinner biofilm formation on gold membranes, the electrical wires touched not only the biofilm, but also the membrane, bypassing the biofilm and leading to imprecise measurements.

    Approximate biofilm conductivity is therefore probably wrong for the gold membrane. However, it is interesting to notice that the biofilm conductivity measured for the bare alumina oxyde, PEDOT:Ts-coated one and PEDOT:PSS-coated give more or less the same value, suggesting that with more measurements, adapted equipement and better methods it would be indeed possible to measure the biofilm's conductivity with our PDMS well chips.