Team:UCopenhagen/Notebook/Week 26







Week 26 (24th-30th of June)

25th of June

1. Competent cells

Team members: Benedicte and Swenja

The use of competent cells can be critical for the success of cloning applications including the transformation of our E. coli cells. Handling of the cells were carried out under the sterile bench

Materials
  • Petri dishes with LB-Agar
  • Falcon tube with 15 ml LB media
  • E. coli culture (Seed stock of WT E coli. from -80 °C freezer can be used)
  • Ice
  • TSS buffer (cold 4 °C)
    • 5g of Polyethylenglycol
    • 0.30 MgCl2*6H2O
    • 2.5 ml DMSO
    • Fill up with LB media to 50 ml
Procedure
  1. 15 ml of LB media was inoculated with 1 ml of E. coli cells and incubated at 37 °C in R141.
  2. The next day, the culture was diluted x100 by adding 0.5 ml of the seed culture to a sterile 250 ml conical flask (R146) containing 50 ml LB media, and subsequently grown to an OD600 of 0.4-0.5. (Then keep on ice). The track of OD with time is mentioned in the table below.
  3. 20 Eppendorf tubes were put on ice and it was made sure that the TSS is chilled (4 °C) in the fridge.
  4. The culture was evenly split up into two Falcon tubes (we got around 2x 24 ml) and incubated for 10 min on ice. All the subsequent steps should be carried out at 4 °C and the cells should be kept on ice wherever possible:
  5. The centrifuge needs to be cooled down to 4 °C 30 min before use!!
  6. Centrifuge the Falcon tubes for 10 min at 3000 rpm and 4 °C.
  7. The supernatant was discarded (if needed carefully remove remaining media with pipette) and the pellet resuspended in TSS buffer (10 % of the culture in Falkon tube before centrifugation). (Higher concentration of cells (2-3x) are found to enhance efficiency, maybe try resuspending in 2-3 % TSS if we have problems with growth again).
  8. We added 20 aliquots of 100 µl into the cooled eppendorf tubes and froze them at -80 °C in R139.
Data
Time Diluted culture left to grow to OD 0.4-0.5 Blank (Only LB media)
13:30 0.032 nm 0.000
14:30 0.067 nm 0.000
15:30 0.408 nm 0.000

2. Normal PCR amplification: X7-PFU Polymerase

Team members: Benedicte and Swenja

To start the USER ligation all over for samples 1 (GPER), 2 (XLHCGR) , 11 (GPER-Linker) and 12 (XLHGCR-Linker), we amplified the original fragments from IDT by PCR. To amplify the gene sequences for sample 1, 2, 11 and 12 (number as labelled on 22nd May 2019) ( For samples 11 and 12, the GPER and XLHCGR fragments were used, respectively. The Linkers are attached to the reverse primers.)

Materials
Materials Quantity (μL)
10X X7 PCR buffer 5
dNTPs 4
F-primer (10 μM) 2.5
R-primer (10 μM) 2.5
Template 0.5
X7-Polymerase (Add last) 0.5
mQ Water ?
Total 50

Procedure

  1. Noted the number of samples to be PCR amplified and calculated the quantities of each material to be added for the master-mix. It should be noted that the amount of water added is to make the total solution of 50 μL.
  2. The mastermix for PCR included the buffer, dNTPs, and water. Mixed the total quantity required based on the number of PCR amplification samples.
  3. Then, we pipette out the individual quantities of Master-mix into each labelled PCR tube.
  4. Now added the primers specific to each PCR sample and the template specific for each PCR sample. Finally we added the X7 polymerase in the end and maintained the sample cold.
  5. Gently mix the final sample, vortex before setting up the PCR machine.
  6. Also, prepare a negative control which includes the entire mix except for the template.
  7. Put the samples into the machine and select the program based on the requirement finally start the program.
  8. PCR program 30 cycles of:
    1. 98 degree celsius: 30 seconds
    2. 98 degree celsius: 20 seconds
    3. 55 degree celsius: 25 seconds
    4. 72 degree celsius: 2 minutes and 30 seconds
    5. 72 degree celsius: 5 minutes
    6. 12 degree celsius: Infinity

Data

SERIAL NO. FRAGMENT NUCLEOTIDE FRAGMENT F-PRIMER R-PRIMER
1 GPER GPER GPER-F GPER-R
2 XLHCGR XLHCGR XLHCGR-F XLHCGR-R
11 GPER-linker GPER GPER-F GPER-linker-R
12 XLHCGR-linker XLHCGR XLHCGR-F XLHCGR-linker-R
Results

In the experimental setup, the amplified sample numbers were labelled such and were arranged serially for further gel electrophoresis.

26th of June

1. Competent cells

Team members: Signe and Swenja

The use of competent cells can be critical for the success of cloning applications including the transformation of our E. coli cells. Handling of the cells were carried out under the sterile bench

Materials
  • Petri dishes with LB-Agar
  • Falcon tube with 15 ml LB media
  • E. coli culture (Seed stock of WT E coli. from -80 °C freezer can be used)
  • Ice
  • TSS buffer (cold 4 °C)
    • 5g of Polyethylenglycol
    • 0.30 MgCl2*6H2O
    • 2.5 ml DMSO
    • Fill up with LB media to 50 ml
Procedure
  1. 15 ml of LB media was inoculated with 1 ml of E. coli cells and incubated at 37 °C in R141.
  2. The next day, the culture was diluted x100 by adding 0.5 ml of the seed culture to a sterile 250 ml conical flask (R146) containing 50 ml LB media, and subsequently grown to an OD600 of 0.4-0.5. (Then keep on ice). The track of OD with time is mentioned in the table below.
  3. 20 Eppendorf tubes were put on ice and it was made sure that the TSS is chilled (4 °C) in the fridge.
  4. The culture was evenly split up into two Falcon tubes (we got around 2x 24 ml) and incubated for 10 min on ice. All the subsequent steps should be carried out at 4 °C and the cells should be kept on ice wherever possible:
  5. The centrifuge needs to be cooled down to 4 °C 30 min before use!!
  6. Centrifuge the Falcon tubes for 10 min at 3000 rpm and 4 °C.
  7. The supernatant was discarded (if needed carefully remove remaining media with pipette) and the pellet resuspended in TSS buffer (10 % of the culture in Falkon tube before centrifugation). (Higher concentration of cells (2-3x) are found to enhance efficiency, maybe try resuspending in 2-3 % TSS if we have problems with growth again).
  8. We added 20 aliquots of 100 µl into the cooled eppendorf tubes and froze them at -80 °C in R139.
Data
Time Diluted culture left to grow to OD 0.4-0.5 Blank (Only LB media)
13:30 0.032 nm 0.000
14:30 0.067 nm 0.000
15:30 0.408 nm 0.000

Gel electrophoresis for sample 1, 11, 2 and 12

Team members: Signe and Swenja

A gel was run with the following samples were carried out: 1, 11, 2 and 12. The corresponding sample names are mentioned below in the table.

Materials
  • Agarose
  • 1X TAE-buffer
  • Loading dye
  • DNA ladder
  • PCR tubes
  • Ice box
Procedure
  1. To prepare the gel
    1. 1% agarose gel was taken from the common stock in electrophoresis room
    2. The Agarose-TAE buffer solution was poured into the casting tray. Please label the cast with iGEM after adding the comb to the solution.
  2. Setting up the electrophoresis chamber
    1. Solidified gel was placed on the electrophoresis chamber filled with TAE buffer
    2. To prepare the sample to be loaded, mixed 4 μL of each sample was taken into a new PCR tube and 1 μL of loading dye was added to each sample.
    3. To load the wells, the sixth well was filled with the 1 kb DNA ladder while wells 1 to 4 were loaded with the PCR samples (1, 2, 11 and 12) while 5 was loaded with negative control.
  3. Finally the electrophoresis setup was put to 100 V in the electric chamber and was run for 25 mins.
Data
Sample no. Fragment
1 GPER
2 XLHCGR
11 GPER-Linker
12 XLHCGR-Linker
Results
The gel was loaded in the following order:
  • Well 1: sample 1
  • Well 2: sample 11
  • Well 3: sample 2
  • Well 4: sample 12
  • Well 5: negative control
  • Well 6: 1 kb ladder

2. DNA purification for sample 1, 2, 11 and 12

Team members: Signe and Swenja

We did PCR purification on the following fragments: 1, 2, 11 and 12. The purification was done using the PCR clean up kit.

Materials
  • Omega PCR clean up kit (Taken from the Gel electrophoresis room).
Procedure
  1. 40 microlitres of all the PCR samples as mentioned in the Table below were transferred into a 1.5 mL microcentrifuge tube.
  2. 200 microlitres of the CP buffer was added.
  3. The samples were vortexed and centrifuged briefly.
  4. HiBind DNA mini columns were inserted into a 2 mL collection tube.
  5. The samples from step 3 were added to the HiBind DNA mini column.
  6. Centrifugation was carried out at 13000 g for 60 seconds at room temperature. The filtrate was discarded and the collection tubes were reused.
  7. 700 microlitres of DNA wash buffer diluted with 100% ethanol was added. Centrifugation was carried out at maximum speed for 60 seconds. The filtrate was discarded and the collection tubes were reused.
  8. Step 7 was repeated again for a second DNA wash buffer step.
  9. The empty HiBind DNA mini columns were centrifuged at maximum speed for 2 minutes to dry the column. This step was critical to remove the trace ethanol.
  10. The HiBind DNA mini column was transferred into a new 1.5 mL microcentrifuge tube.
  11. 50 microlitres of elution buffer was directly added to the center of the column matrix. It was kept at room temperature for 2 minutes and was centrifuged at maximum speed for 60 seconds.
  12. The DNA was stored at -20⁰C.
Data
Table showing sample no., fragment and nanodrop concentration. The nanodrop concentration was measured on the 27th of june and not on the 26th as stated above. The nanodrop concentration can also be found in the lab image folder.
Sampel no. Fragments A260/A280 A260/230 Concentration (ng/μl)
1 GPER 1.82 1.86 2.09 2.17 97.0 91.8
2 XLHCGR 1.85 1.82 2.21 2.10 87.6 88.6
11 GPER-Linker 1.80 2.08 85.3
12 XLHCGR-Linker 1.79 2.06 78.4
Results

The purified DNA was labelled and stored at -20⁰C. The results from the nanodrop looked decent.

27th of June

1. E.coli transformation samples 1,2,4 and 6

Team members: Benedicte and Swenja

After preparing our competent cells, we were ready to perform transformation of our competent E.coli cells with the USER ligated samples 1, 2, 4 and 6 from 14th June 2019. We followed the same protocol as earlier, but with a longer incubation period, due to a low number of colonies in the previous experiment.

In the following we will continue to refer to the samples as 1, 2, 4, 6
1 2 4 6
GPER + pCCW12 XLHGCR + pCCW12 XLHGCR-Linker + sfGFP + pCCW12 GPA1-Gαs + pPGK1

Materials

  • Competent E.coli cells
  • USER reacted samples
  • Ice box
  • LB media

Procedure

  1. The competent E.coli cells (8 eppendorfs with 50 μl each) were obtained from R-139, from the freezer (-80 °C).
  2. These cells were moved to ice and then thawed.
  3. 3 μl of each USER sample and 50 μl competent cells were added to 1.5ml eppendorf tube.
  4. The tubes were kept on ice for 5 minutes, followed by a 30-45 seconds heat shock at 42 °C and was again kept on ice briefly.
  5. 1 ml of LB media was added to all the tubes, which were then incubated at 37 °C for 1 hour.
  6. Centrifugation was carried out at 8000 g for 60 seconds and 900 μl of the supernatant was discarded.
  7. With the remaining 100 μl media, the cells were resuspended and then added on to the agar plates and were spread evenly.
    1. This step was performed at the sterile bench, to prevent contamination.
    2. The plates were named S1, S2, S4, S6 contain cells with their corresponding USER samples.
    3. The plates were then left inside the incubator overnight to allow growth of colonies.
    Data
    1 X3A pCCW12 GPER
    2 X3A pCCW12 XLHCGR
    4 X3A pCCW1 XLHCGR-linker + sfGFP
    6 Ass2A pPGK1 GPA1-Gαs
    Results

    After incubation at 37 C overnight, colonies were present in the following which were further advanced for colony PCR dated 28th June 2019.

    1. S2: Colonies 1 to 6 were chosen for the PCR
    2. S1 and S4: There were only 3 colonies on each of the plates, so all 6 colonies were chosen for PCR.
    3. S6: There were no colonies on the plates.

    2. Making our USER vector stock

    Team members: Anett

    The goal for the afternoon was to grow E.coli cells (that have plasmids X3A, X3C, BAss2, Ass2A, Ass2B, Ass2C) in LB and on plates containing Carbenicillin ON. Thus, the next day we can make our own glycerol stock from them, we can purify the USER vectors and digest it. We worked with 6 glycerol stock tubes from Nat which contained E.coli with: X3A, X3C, BAss2, Ass2A, Ass2B, Ass2C.

    Materials
    • Nat’s glycerol stock tubes: X3A, X3C, BASS2, ASS2A, ASS2B, ASS2C.
    • LB + Carbenicillin and an 50 ml tube
    • Plates with Carbenicillin
    • Sterile loops
    • Parafilm
    • Icebox+ice
    Procedure
    1. Ask for Nat’s glycerol stock: X3A, X3C, BASS2, ASS2A, ASS2B, ASS2C. Keep them on ice! Put them back to -80°C as soon as possible.
    2. Go to the sterile bench, pour 5-10 ml LB + Carbenicillin into 50 ml falcon tubes
    3. Divide the plates with Carbenicillin resistance into 3-4 parts with a marker.
    4. Dip a sterile loop into the glycerol stock and plate it then dip it into the 50 ml tubes as well. Parafilm it.
    5. Put it to 37 °C ON.

    28th of June

    1. Colony PCR for samples 1,2,4 and 6

    Team members: Jonas and Hitesh

    AIM: Colony PCR was carried out for the samples S1, S2, S4 and S6

    GPER + pCCW12 (S1) XLHGCR + pCCW12 (S2) XLHGCR-Linker + sfGFP + pCCW12 (S4) GPA1-Gαs + pPGK1 (S6)

    S2: Colonies 1 to 6 were chosen for the PCR

    S1 and S4: There were only 3 colonies on each of the plates, so all 6 colonies were chosen for PCR.

    S6: There were no colonies on the plates.

    Materials
    • 10X X7 PCR buffer
    • X7 polymerase
    • F-primer
    • R-primer
    • dNTP
    • Template: Colonies from overnight plates

    Procedure

    1. Standard colony PCR protocol was followed dated 28th May 2019.
    Results

    The amplified product was further advanced for gel electrophoresis.

    2. Competent cell test

    Team members: Hitesh, Jonas, Anett and Swenja

    Aim: To test the efficiency of the competent cells (E.coli) prepared.

    Materials
    • Competent cell test kit
    Procedure
    1. The kit includes two vials of dried-down purified plasmid DNA from labelled as Biobrick BBa_J04450 (RFP construct) in plasmid backbone pSB1C3 in the iGEM registry. The plasmid backbone contains the gene for chloramphenicol resistance which we forgot to take note of when we performed the experiment.
    2. When resuspended with 50 µL dH2O, the vials will result in different concentrations: 100 pg/µL, 10 pg/µL. Perform transformations with each of these to determine how efficient your competent cells are.
    3. Standard E.coli transformation protocol dated 27th May 2019 can be used to test the competent cells.
    4. The transformed E.coli cells (10 pg/µL and 100 pg/µL) were plated and incubated overnight.

    We finished what Hitesh and Jonas started in the morning. Thus, we plated E.coli cells to check if the competent cells made on the 27th of June are really competent. The test was accomplished in the morning with the kit that we got from iGEM HQ. We put the plates (no antibiotic) to 37 °C incubator ON. Jonas took them out next morning (28th June). Result: There were too many colonies (almost like a lawn formation). This is because there was no antibiotic added to the agar plates, so even the non-transformed E.coli grew on the plates. But the plasmid backbone contains chloramphenicol resistance gene, so chloramphenicol is to be added to the agar plates next time.

    3. Gel electrophoresis for samples 1,2,3 and 6

    Team members: Swenja and Anett

    A gel was run with the following samples were carried out: 1, 2, 4 and 6 from the morning of the 28th June. The corresponding sample names are mentioned below in the table.

    GPER + pCCW12 (S1) XLHGCR + pCCW12 (S2) XLHGCR-Linker + sfGFP + pCCW12 (S4) GPA1-Gαs + pPGK1 (S6)

    Materials
    • Agarose
    • 1X TAE-buffer
    • Loading dye
    • DNA ladder
    • PCR tubes
    • Ice box
    Procedure
    1. To prepare the gel
      1. 1% agarose gel was taken from the common stock in electrophoresis room
      2. The Agarose-TAE buffer solution was poured into the casting tray. Please label the cast with iGEM after adding the comb to the solution.
    2. Setting up the electrophoresis chamber
      1. Solidified gel was placed on the electrophoresis chamber filled with TAE buffer
      2. To prepare the sample to be loaded, mixed 4 μL of each sample was taken into a new PCR tube and 1 μL of loading dye was added to each sample.
      3. To load the wells, the format followed is mentioned below.
      4. Finally the electrophoresis setup was put to 100 V in the electric chamber and was run for 25 mins.
    Data

    We also run the gel from the colony PCR that Hitesh and Jonas did in the morning. Unfortunately, the gel was empty. Gel: Ladder, Sample1-Colony 1, Sample1-Colony 2, Sample1-Colony 3, Sample1- Negative control, Sample4- Colony 1, Sample4- Colony 2, Sample4-Colony 3, Sample4- Negative control, Sample2-Colony 1, Sample2-Colony 2, Sample2-Colony 3, Sample2-Colony 4, Sample2-Colony 5, Sample2-Colony 6, Sample2- Negative control, Ladder.

    Results

    Unfortunately, the gel was empty.

    4. Plasmid purification

    Team members: Swenja and Anett

    We finished the plasmid DNA purification from the E.coli samples from 27th June as the next step to get our USER vector stock. We followed the protocol in the plasmid DNA mini kit and for the elution we used 50ul elution buffer. We run the gel with the samples: Ladder, X3A, BASS2, X3C, ASS2A, ASS2B, ASS2C.

    Materials
    • E.Z.N.A. Plasmid DNA Mini Kit I.
    Procedure
    1. We took out the tubes from the 37 °C incubator. There was growth in all of them.
    2. We found the miniprep kit in R132: E.Z.N.A. Plasmid DNA Mini Kit I.
    3. We vortexed the liquid cultures and pipetted 2 ml into labelled eppendorf tubes. We put the rest (labelled) into the fridge in a plastic bag.
    4. We centrifuged at 10 000 g for 1 min (mistake: we did it at 12 °C instead of room temperature.
    5. We poured out the culture media and kept the pellet.
    6. We added 250 µl Sol I, vortexed the samples, transferred the suspension into new microcentrifuge tubes.
    7. We added 250 µl Sol II, inverted the samples and waited for 2-3 minutes (timing is important!)
    8. We added 350 µl Sol III, inverted the tubes.
    9. We centrifuged the samples at maximum speed for 10 minute. White pellet was formed.
    10. We skipped the optional column equilibration (because of the lack of some solutions).
    11. We transferred the supernatants into columns in 2 ml collection tubes.
    12. We centrifuged it for 1 min at maximum speed, discarded the filtrate.
    13. We skipped the 8. and 9. Step of the protocol (we did not have HBC solution a researcher recommend us to try it without).
    14. We added 700 µl DNA Wash Buffer, centrifuged it for 30d, discarded the filtrate.
    15. We repeated the previous step again.
    16. We centrifuged the empty column for 2 min at maximum speed to dry the column.
    17. We transferred the column into new microcentrifuge tubes.
    18. We added 50 µl Elution Buffer, let it sit for 1 minute and then we centrifuged it for 1 min at maximum speed.
    19. We did not get any elution (probably because we had to use not the proper column for our experiments). We decided to elute it again (maximum speed but for 5 minutes this time). We got elution!
    20. We ran a gel with 4 µl sample + 1 µl gel red, 5 µl ladder. 100 V for about 25 min. The rest of the sample is in the freezer (label for example: 1. 30-05-2019 Purified pl. DNA.
    Data
    Revised Tube numbers SAMPLE (Plate number previously) Positive colonies VECTOR (used to transform E coli in that plate)
    1 S1 Colony 2 X3A+pCCW12+GPER
    2 S2 Colony 1 X3A+pCCW12+XLHCGR
    3 S5 Colony 1 Ass 2A+pPGK1+GPA1-Gαi
    4 S5 Colony 2 Ass 2A+pPGK1+GPA1-Gαi
    5 S6 Colony 4 Ass 2A+pPGK1+GPA1-Gαs
    6 S6 Colony 5 Ass 2A+pPGK1+GPA1-Gαs
    7 S7 Colony 1 Ass 2B+pRET2+STE12
    8 S7 Colony 2 Ass 2B+pRET2+STE12
    9 S8 Colony 4 X3C+pFIG1+ZsGreen
    10 S8 Colony 5 X3C+pFIG1+ZsGreen
    Ladder, X3A, BASS2, X3C, ASS2A, ASS2B, ASS2C.
    The list of vector backbone samples that were purified, their respective absorbance ratios and the Nanodrop concentration are calculated and mentioned here dated 1st July 2019 by Ojas.
    Sample name A260/280 A260/230 ng/µL
    X3A 1.83 2.12 113.2
    Ass2A 1.80 2.01 58.4
    Ass2B 1.85 1.91 40.3
    Ass2C 1.81 2.04 51.2
    BAss2 1.87 2.20 74.9
    X3C 1.84 2.14 71.4
    Results

    Vector backbone purification performed, gel electrophoresis run for the same purified samples and then finally nanodrop concentrations measured.

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