Team:UCopenhagen/Notebook/Week 30







Week 30 (22nd-28th of July)

22nd of July

1. Yeast transformation

Team members: Jonas and Ojas

Yeast transformation was not successful for the first time and therefore we made new transformations for a few 5 assembler systems and a few for 3 assembler system.

Materials
  • Book Yeast lab bench at room T-155 in advance
  • O/N culture
  • Glu-U +Agar media
  • Cuvettes and spectrophotometer for OD value check
  • Falcon tubes
  • Plasmid samples for all modules
  • Plasmid samples for all modules
  • NotI enzyme
  • CutSmart Buffer
Procedure
  1. The O/N yeast culture grown in liquid YPD media is diluted 1:10 (100 µl culture + 900 µl YPD media only)
  2. OD of the dilution is measured at 600 nm with pure YPD as standard blank
    1. OD measurement in room R251: OD=0,655. Since there is a linear correlation between the diluted and undiluted O/N culture it is calculated that the OD of the undiluted O/N is 6,55.
  3. The undiluted O/N culture is now diluted to achieve an OD of 0,25 for a total volume of 25 mL based on calculation on the OD of the diluted ON culture.
    1. Calculations: C1*V1=C2*V2

      V1=(0,25*25 ml)/6,55=0,9541 ml≈0,95 ml
    2. Therefore, Dilution: 0,95 ml of ON culture + 24,05 ml of YPD was prepared.
  4. The 25 ml ON dilution (OD 0.25) was then kept in an incubator at 30 °C and 150 RPM for approximately 4 hours. Note the time when you put the yeast into the incubator, keep a track of that. (the idea is that the O/N culture of yeast reaches a saturation point where almost all cells are at the same phase and now we prepare a required dilution let the yeast undergone 2 fresh cell divisions)
  5. Prepare the GMO Yeast lab bench at room T-155 in advance and book it on the schedule by mentioning iGEM.
  6. During the 4 hour incubation we prepared the plates (for transformed yeast plating) with GLU -U media (Glucose medium without uracil which would be the selection marker). They were prepared in a sterile bench using already prepared media (plates should be thicker than the plates used to grow E. coli).
  7. At the same time we had to do the ligation of different modules that are in different plasmids in order to transform the yeast with desired module only. To do so, the concentration, volume and amount of DNA was calculated for all the DNA samples which was going to be used.
    Sample Concentration (ng/µl)
    1.1 X3A-pCCW12-GPER 164.45
    2.2 X3A-pCCW12-GPER-sfGFP 219.65
    3.1 X3A-pCCW12-XLHCGR-sfGFP 204.75
    Ass2A-pGK1-Gαi 118.8
    Ass2B-pRET2-STE12 114.05
    X3C-pFIG1-ZsGreen 122.05
    X3C 256.4
    BAss2 248.3
    Ass2C 137.4
  8. he uL of DNA we would require were calculated based on the assembler system we are using that is for a 5 assembler system we calculated uL corresponding to 1000 ng of X3A-pCCW12-GPER while 1500ng of all the other plasmids (each plasmid sample) while for 3-assembler system we calculated uL corresponding to 600 ng of X3A-pCCW12-GPER-sfGFP and X3A-pCCW12-XLHCGR-sfGFP respectively while 900 ng of all the other plasmids (each plasmid sample) and the calculated uL are mentioned in the table below.
    Sample no. X3A module µL Ass2A module µL Ass2B module µL Ass2C module µL X3C module µL Total (µL)
    1 X3A-pCCW12-GPER 6.08 Ass2A-pPGK1-GPAGαi 12.63 Ass2B-pRET2-STE12 13.15 Ass2C 10.91 X3C-pFIG1-ZsGreen 12.29 56.1
    2 X3A-pCCW12-GPER-sfGFP 2.73 BAss2 - - - - 3.62 X3C 3.51 10.88
    3 X3A-pCCW12-XLHCGR-sfGFP 2.93 BAss2 - - - - 3.62 X3C 3.51 11.08
    4 X3A-pCCW12-GPER 6.08 Ass2A-pPGK1-GPAGαi 12.63 Ass2B-pRET2-STE12 13.15 Ass2C 10.91 X3C-pFIG1-ZsGreen 12.29 56.1
  9. The DNA mixes/samples has to be digested by NotI and left in the buffer for ligation. Therefore the total volume of the digestion reactions has to be calculated along with the amount of buffer needed in each reaction. The buffer volume should be 1/10th of the total volume therefore a calculation was made. For ex. Let volume of buffer to be added be x. Now, x/x+Volume of plasmid sample mix should be equal to 1/10. The further calculated volumes are mentioned in the table below. The enzyme is added as the last component:
    Sample Total volume (µl) Volume of buffer (µl) Volume of enzyme (Not1) (µl)
    1 56.1 6.23 1.5
    2 10.88 1.21 1.5
    3 11.08 1.23 1
    4 56.1 6.23 1.5
  10. After the enzyme has been added quickly votex the samples slightly and thereafter spin the samples down briefly.
  11. The digestion samples then have to incubate at 37 °C for 2 hours
  12. Inactivate the enzyme at 65 °C for 20 min and thereafter set the next step at 12 °C indefinitely
  13. Some salmon sperm DNA was denatured by putting it on a heat block at 99 °C
  14. The OD was measured to 0,7 which was too low, so the cultures were incubated for 20 min (in the shaking incubator) to reach the desired OD.
  15. When the desired OD was reached, the cultures ∆5 were transferred from the erlenmeyer flasks to Falcon tubes
  16. These were centrifuged at 4000 rpm for 5 min
  17. The salmon sperm DNA (that will denature by being boiled for at least 5 min) was cooled down quickly by being put on ice so that it would not re-anneal.
  18. The supernatant from the centrifuged Falcon Tubes containing was discarded (this was just poured out)
  19. The pellet was resuspended in 1 mL Lithium acetate (with water)
  20. The resuspension (1 mL) was transferred into an eppendorf tube.
  21. These were centrifuged at 8000 rpm for 1 min and the supernatant was discarded (by pipetting).
  22. The pellet was resuspended in 100 µL Lithium acetate (with water) (Tip: resuspend with big pipette to stress the cells less).
  23. 30 µL of denatured salmon sperm DNA was put into the eppendorf tubes (with the yeast cells and the Lithium acetate) and this was gently mixed.
  24. The PLI was mixed by being inverted (not shaken because the PLI is hydroscopic).
  25. 1 mL of PLI was put into the eppendorf tubes (PLI stabilizes the cells). It was homogenized by pipetting up and down.
  26. The digested and ligated plasmids was transferred to eppendorf tubes.
  27. The eppendorf tubes were labelled 1-8 following the DNA samples. 8 was the negative control.
  28. 200 µL of the mix in eppendorf tubes with the PLI and Lithium acetate and the yeast cells was aliquoted in each of the 8 eppendorf tubes.
  29. The samples were heat shocked at 42 °C for 30 min at 750 rpm.
  30. After being heat shocked, the samples were centrifuged for 1 min at 8000 rpm and the supernatant was discarded.
  31. 1 mL water was added (to wash the cells) and the cells were resuspended by being vortexed (vortexing is faster and less rough on the cells).
  32. They were again centrifuged for 1 min at 8000 rpm and 900 µL of the supernatant was discarded.
  33. The cells were resuspended in the remaining 100 µL and they were mixed gently.
  34. hey were plated (and named according to the DNA samples) and kept in an incubator for 3 days so colonies could grow.
Results

Yeast colony PCR was performed after incubation for 3 days dated 26th July 2019.

23rd July

1. Media and reagent preparation

Team members: Selma and Swenja Materials
  • YPD + Agar
  • Glu-U + Agar
  • 10% NaOH
  • Glu-U liquid media
  • YPD liquid media
Procedure
Results

Left for autoclave at R 218 and collected the next day.

24th July 2019

1. Primer dilution

Team members: Iben and Noël

To dilute the gene fragments and primers

Materials
  • Milli-Q-water
  • Samples primers: U-hTPST1-F, U-hTPST1-R, U-hTPST2-F, U-hTPST2-R and U-HuLHCGR-myc-R.
Procedure
  1. For primer dilution:
    1. Took the samples delivered by the IDT in powder form to dilute in Milli-Q-water and make a final stock solution to 100 μM. To do that, the OD values were given in nM concentrations and amount of water to be added to make a final concentration of 100 μM was calculated as in Table 1.
    2. Further took a part of the Stock solution to make a working solution of 10 μM. To do so, took 10 μL of each stock solution and added 90 μL of water to it.
    2. Primer Sample name OD value (nM) μL of water added
    U-hTPST1-F 31.4 314
    U-hTPST1-R 30.4 304
    U-hTPST2-F 24.7 247
    U-hTPST2-R 34.2 342
    U-HuLHCGR-myc-R 24.7 247

    Table 1 The list of primers and their concentration as supplied by IDT and the amount of water to be added to make a final concentration for the stock solution.

    Results

    Stock solutions stored in the freezer at -20 °C. Working solution arranged to be used also at freezer.

    1. EZNA Plasmid purification

    Team members: Ojas

    The plasmid purification was performed on 12 samples (in total) of E. coli transformed with different plasmids. Of which 1-10 samples were from 29th May 2019 while other 2 samples Sample 11 and 12 were from 6th June 2019. These samples from their respective dates were grown on a fresh LB media + Carbenicillin a night before and then used for plasmid purification. However, in the morning 750 uL of each of these E. coli grown in fresh media were stored in eppendorf tubes to be made into glycerol stock later.

    Materials
    • E.Z.N.A. Plasmid DNA Mini Kit I.

    Procedure

    1. Followed the protocol as described in the kit entirely as it is.

    Data

    Sample No. Sample name
    1 X3A-pCCW12-GPER
    2 X3A-pCCW12-XLHCGR
    3 Ass2A-pGK1-Gαi
    4 Ass2A-pGK1-Gαi
    5 Ass2A-pGK1-Gαs
    6 Ass2A-pGK1-Gαs
    7 Ass2B-pRET2-STE12
    8 Ass2B-pRET2-STE12
    9 X3C-pFIG1-ZsGreen
    10 X3C-pFIG1-ZsGreen
    11 X3A-pCCW12-GPER-sfGFP
    12 X3A-pCCW12-GPER-sfGFP
    Results

    Nanodrop concentrations were measured for the purified plasmids.

    Sample No. Sample name Concentrations (ng/uL)
    1 X3A-pCCW12-GPER 56.8
    2 X3A-pCCW12-XLHCGR 84.1
    3 Ass2A-pGK1-Gαi 74.2
    4 Ass2A-pGK1-Gαi 61.1
    5 Ass2A-pGK1-Gαs 65.2
    6 Ass2A-pGK1-Gαs 56.8
    7 Ass2B-pRET2-STE12 57.2
    8 Ass2B-pRET2-STE12 65.4
    9 X3C-pFIG1-ZsGreen 43.7
    10 X3C-pFIG1-ZsGreen 46.7
    11 X3A-pCCW12-GPER-sfGFP 127.5
    12 X3A-pCCW12-GPER-sfGFP 186.7

    25th July 2019

    1. Sample preparation for sequencing

    Team members: Ojas and Swenja

    The plasmids which had been purified on the 24th of July were to be prepared for sequencing. Unfortunately, the DNA concentrations were very low (confirmed by second NanoDrop measuring), why only samples 11 and 12 were prepared.

    Materials
    • Vector
    • Primers
    • mH2O
    • Templates
    Procedure
    1. To assure that the whole constructs would be synthesised three primers were used for each vector (USER primer of the first gene and two further primers according to list in R148) Note: We used Sanger sequencing which is limited to the sequencing of approximately 700 bp at a time. Therefore an extra primer was used.
    2. For each construct only one sample was prepared for sequencing (Samples 1, 2, 4, 5, 7, 10). Moreover three eppendorf tubes had been prepared per sample, one for each of the three primers. The samples were prepared as follows:

      DNA: µl corresponding to 500 ng DNA

      Primer: 3,3 µl

      mH20: x µl

      Reaching a total of 10 µl
    3. The samples were labelled with sequencing stickers containing QR codes. The stickers consists of two parts, the smaller of which was attached into the lab book in a list next to the corresponding construct and primer. The second part was wrapped around the eppendorf tubes in such a way that the QR code was clearly visible.
    4. The samples were put in a plastic bag (make sure the tubes are closed properly) and inserted into the envelope in the box on top of the shelf that is located in the hallway on the right behind the PCR room.
    5. The samples have been registered on www.ezseq.com.
    Data
    Sample Nr. Sample name Note 1. Primer 2. Primer 3. Primer
    11 X3A-pCCW12-GPER-sfGFP Sample 3, Colony 4 YEA74 YEA81 GPER-F
    12 X3A-pCCW12-GPER-sfGFP Sample 3, Colony 5 YEA74 YEA81 GPER-F
    Results

    The sequencing results were received from macrogen and analyzed by aligning them with the original sequences using SnapGene. The sequences were confirmed to be correct as they didn’t show any mismatches in the regions with high accuracy. However, both of the construct duplicates showed two missing base pairs in the promoter region.

    2. Glycerol stock

    Team members: Ojas and Hitesh

    The transformed E. coli samples; 12 samples (in total) of E. coli transformed with different plasmids. Of which 1-10 samples were from 29th May 2019 while other 2 samples Sample 11 and 12 were from 6th June 2019 were grown overnight in a fresh LB medium with Carbenicillin and stored 750 uL of each dated 24th July 2019 to make glycerol stocks of each. We then made glycerol stock with 700 uL of transformed E. coli grown in liquid LB medium with 300 uL of 50% glycerol solution.

    Materials
    • Transformed E coli colonies in liquid LB + Carbenicillin medium
    • 50% glycerol solution
    Procedure
    1. We made glycerol stock with 700 uL of transformed E. coli grown in liquid LB medium with 300 uL of 50% glycerol solution.
    Data
    Sample No. Sample name
    1 X3A-pCCW12-GPER
    2 X3A-pCCW12-XLHCGR
    3 Ass2A-pGK1-Gαi
    4 Ass2A-pGK1-Gαi
    5 Ass2A-pGK1-Gαs
    6 Ass2A-pGK1-Gαs
    7 Ass2B-pRET2-STE12
    8 Ass2B-pRET2-STE12
    9 X3C-pFIG1-ZsGreen
    10 X3C-pFIG1-ZsGreen
    11 X3A-pCCW12-GPER-sfGFP
    12 X3A-pCCW12-GPER-sfGFP
    Results

    The glycerol stocks were stored in the -80 degrees freezer.

    3. EZNA plasmid purification

    Team members: Ojas

    The plasmid purification was performed on 12 samples (in total) of E. coli transformed with different plasmids. Of which 1-10 samples were from 29th May 2019 while other 2 samples Sample 11 and 12 were from 6th June 2019. These samples from their respective dates were grown on a fresh LB media + Carbenicillin a night before dated 24th July and then used for plasmid purification.

    Materials
    • E.Z.N.A. Plasmid DNA Mini Kit I.
    Procedure
    1. Followed the protocol as described in the kit entirely as it is.
    Data
    Sample No. Sample name
    1 X3A-pCCW12-GPER
    2 X3A-pCCW12-XLHCGR
    3 Ass2A-pGK1-Gαi
    4 Ass2A-pGK1-Gαi
    5 Ass2A-pGK1-Gαs
    6 Ass2A-pGK1-Gαs
    7 Ass2B-pRET2-STE12
    8 Ass2B-pRET2-STE12
    9 X3C-pFIG1-ZsGreen
    10 X3C-pFIG1-ZsGreen
    11 X3A-pCCW12-GPER-sfGFP
    12 X3A-pCCW12-GPER-sfGFP
    Results

    Nanodrop concentrations were measured for the purified plasmids.

    Sample No. Sample name Concentrations (ng/uL)
    1 X3A-pCCW12-GPER 158.1
    2 X3A-pCCW12-XLHCGR 152.3
    3 Ass2A-pGK1-Gαi 135.9
    4 Ass2A-pGK1-Gαi 104.3
    5 Ass2A-pGK1-Gαs 94.4
    6 Ass2A-pGK1-Gαs 79.0
    7 Ass2B-pRET2-STE12 94.4
    8 Ass2B-pRET2-STE12 86.5
    9 X3C-pFIG1-ZsGreen 49
    10 X3C-pFIG1-ZsGreen 73.8
    11 X3A-pCCW12-GPER-sfGFP 155.1
    12 X3A-pCCW12-GPER-sfGFP 157.9

    4. Gel electrophoresis of old PCR products

    Team members: Swenja and Ojas

    Gel electrophoresis was performed for 4 PCR amplified products dated 26th June, 4 PCR amplified products dated 1st July and 8 PCR amplified products dated 12th July.

    Materials
    • 1% agarose
    • 1x TAE buffer1 kb DNA ladder
    • Loading dye
    • PCR tubes
    • Ice
    • Templates
    Procedure
    1. To prepare the gel 1% agarose was taken from the common stock in electrophoresis room.
    2. The agarose-TAE buffer solution was poured into casting trays with 30 and 23 wells, respectively. (Please label the cast with iGEM after adding the comb to the solution.)
    3. Solidified gel was placed on the electrophoresis chamber filled with TAE buffer.
    4. To prepare the sample to be loaded, mixed 5 μL of each sample was taken into a new PCR tube and 1 μL of loading dye was added to each sample.
    5. The wells were loaded according to the images below. A 1 kb DNA ladder was added before/after each set of samples. Finally the electrophoresis setup was put to 100 V in the electric chamber and was run for 50-60 mins (big electrophoresis chamber).
    Data

    The gel were loaded in the following order:

    1. Ladder
    2. GPER
    3. XLHCGR
    4. GPER-Li
    5. XLHCGR-Li
    6. Ladder
    7. GPER
    8. XLHCGR
    9. GPER-Li
    10. XLHCGR-Li
    11. Ladder
    12. pCCW12
    13. pPGK1
    14. pRET2
    15. pFIG1
    16. GPA1-Gai
    17. GPA1-Gas
    18. STE12
    19. Zs-Green
    Results

    The image of the gel:

    5. E.coli transformation with Ass2A-pPGK1-GPA1-GaTHs

    Team members: Ojas

    We made a USER ligation sample for Ass2A-pPGK1-GPA1-Gas and then transformed E. coli competent cells with the USER assembled plasmid.

    Materials
    • Competent E.coli cells
    • USER reacted samples
    • Ice box
    • LB media
    Procedure
    1. The competent E.coli cells (1 eppendorfs with 50 μl) 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 were added to their respective 50 μl competent cells containing tubes.
    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 for 10 minutes.
    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.
    8. The plate was labelled Ass2A-pPGK1-GPA1-Gas.
    9. All the plates were finally sealed with parafilm and left inside the incubator to allow growth of colonies.
    Results

    We got the colonies when checked the next day. And the colony PCR was performed on 29th July 2019

    26th July 2019

    1. Colony PCR of transformed yeast from 22nd July

    Team members: Jonas and Hitesh

    Colony PCR I of transformed yeast from 22nd july 2019

    Materials
    • NaOH solution
    • MangoMix master mix solution
    • F1-primer
    • F2-primer
    • R1-primer
    • Template: Colonies from overnight plates
    Procedure
    1. Eight colonies from the transformation (22nd of July) were picked from each plate and each colony was put into 20 µl of 20 mM NaOH. The colonies were marked with a dot and a number from 1-8 on the plates.
    2. The NaOH solution (hereafter referred to as dirtboil) with the yeast were then boiled for 10 at 99 °C.
    3. The master mix for the PCR reaction was prepared as per the table below. There was prepared enough mastermix for 33 reactions including a negative control.
      1. The negative control did not contain template
    4. The master mix was aliquoted into 33 PCR tubes afterwhich 1 µl of dirtboil was added to each PCR tube (achieving a total volume of 10 µl).
    5. Hereafter the PCR tubes were put in the thermocycler using the following program (b-e where run for 35 cycles):
      1. 96 °C: 30 seconds
      2. 96 °C: 20 seconds
      3. 55 °C: 25 seconds
      4. 72 °C: 90 seconds (1 min pr. kb)
      5. 72 °C: 5 minutes
      6. 12 °C: Infinity
    6. Thereafter the samples were put into the -20 °C freezer.
    Materials Quantity (μL)
    NaOH solution 20
    2x MangoMix (commercial mastermix solution) 5
    F1-primer 0.5
    F2-primer 0.5
    R1-primer 0.5
    milli-Q-water 2.5
    In total 9

    The composition of materials to be used per colony PCR amplification.

    Results

About Us

We are Ovulaid: a team of 13 students from the University of Copenhagen working on a novel ovulation detection system, using synthetic biology.

Keep in Touch

iGEM Team Copenhagen

iGEM_Copenhagen

iGEM_Copenhagen

UCPH.IGEM2019@gmail.com

Address

University of Copenhagen

Thorvaldsensvej 40, Frederiksberg C

Denmark