Team:UCopenhagen/Notebook/Week 22







Week 22 (27th of May-2nd of June)

27th of May

1. Nanodrop

Team members: Benedicte, Ojas and Hitesh

To quantify and assess the purity of purified PCR products

Materials
  • PCR products
  • Nanodrop spectrophotometer
Procedure
  1. Purified DNA samples were taken from the freezer and thawed for a while.
  2. The samples were taken to R-133 to use the nanodrop spectrophotometer.
  3. On the desktop, Nanodrop 1000 3.8.1 software was opened.
  4. Once the program was opened, the nucleic acid tab was selected, which analyses the DNA and RNA.
  5. The nanodrop arm was opened and the nanodrop sensor was cleaned with 1 microlitre water.
  6. After cleaning the nanodrop, 1 microlitre of water was again placed on the nanodrop sensor and the arm was lowered and Blank button was pressed on the dialogue box to set the blank.
  7. Once the blank has been set, the arm was lifted and the sensor was wiped with a tissue paper.
  8. Now 1 microlitre of the sample was placed on the sensor and the measure button was clicked on the dialogue box.
  9. Out of the generated values, A 260/280 ratio and the concentration in ng/μl for the sample was noted.
  10. Steps 8 and 9 were repeated for all the 13 samples.
  11. At the end, the data report was saved on the desktop and was uploaded in the Lab folder in our iGEM drive.
Data
The table shows the A260/280 ratio and the concentrations in ng/μl for all the purified products
S. No. SAMPLE NO. NUCLEOTIDE FRAGMENT A260/230 Concentration (ng/μl)
1 1 GPER 1.98 39.7
2 2 XLHCGR 1.93 42.1
3 3 pCCW12 1.96 31.3
4 4 GPA1-Gαi 1.87 65.3
5 5 GPA1-Gαs 2.06 44.8
6 6 pPGK1 2.13 18.4
7 7 STE12 1.88 48.5
8 8 pRET2 1.83 33.1
9 9 ZsGreen 1.92 75.9
10 10 pFIG1 1.88 68.4
11 11 GPER-linker 1.85 227.8
12 12 XLHCGR-linker 1.86 99.1
13 13 sfGFP 1.86 104.4
Conclusion

The purity of the DNA samples was assessed and the concentrations were tabulated.

2. Adapted user reaction (ligation)

Team members: Ojas

The purified sample of each DNA fragment was then used with an adapted USER ligation reaction protocol.

Materials
  • DpnI enzyme
  • 10X USER enzyme
  • 10X CutSmart Buffer
  • Promoter fragments
  • Gene fragments
  • Linearised vector backbones
Procedure
  1. Make a total of 10 μL digestion reaction first for each of the sample 1, 2, 11, 12, 4, 5, 7, and 9 as mentioned in the Table below. The number of reaction samples depend upon the number of genes to be integrated, each gene with its specific promoter goes into one of the vector backbones and therefore must be digested together.
  2. The final digestion mix is put into the Thermocycler PCR machine at;
    1. 37 °C for 30 mins
    2. 80 °C for 20 mins
    3. 25 °C for infinity
  3. Each sample (promoter + gene) is cloned into a different vector and therefore once the digested product is ready, 1 μL of linearised vector backbone is added onto each 10 μL reaction digested product samples 1, 2, 11, 12, 4, 5, 7, and 9 based on their respective backbone as mentioned in the table below. The final mix of linearised vector backbone added to the digested product sample was then put in the thermocycler PCR machine for step-down cooling for the ligation reaction to occur at;
    1. 37 °C for 10 mins
    2. 25 °C for 10 mins
    3. 20 °C for 10 mins
    4. 12 °C infinity
  4. We use a 5-assembler system for genomic integration of the genes.
    1. X3A: Promoter pCCW12 with receptor
    2. Ass 2A: Promoter pPGK1 with GPA1-Gαi, GPA1-Gαs
    3. Ass 2B: Promoter pRET2 with Transcription factor STE12
    4. Ass 2C: Empty for our system
    5. X3C: Promoter pFIG1 with ZsGreen reporter gene
  5. The vector backbones are linearised before mixing with the promoter and gene fragment mix. The vector backbones of the USER cassette are digested with restriction enzymes AsiSI and NbBsmI that cut at specific sites in the backbone that linearises the backbone and creates sticky ends that are specific for our digested sequences (promoter + gene fragment) to bind.
Data
Vector backbone, promoter and the respective gene clubbed together in the same sample for digestion with enzyme and then ligation as mentioned with the sample number 1, 2, 11, 12, 4, 5, 7, and 9. The ligation product when transformed to E. coli was renamed from Serial number 1-8
SERIAL NUMBER REVISED EXPERIMENT SERIAL NUMBER FORMERLY VECTOR PROMOTER 1μL GENE FRAGMENT 1μL
1 1 X3A pCCW12 GPER
2 2 X3A pCCW12 XLHCGR
3 11 X3A pCCW12 GPER-linker + sfGFP
4 12 X3A pCCW12 XLHCGR-linker + sfGFP
5 4 Ass 2A pPGK1 GPA1-Gαi
6 5 Ass 2A pPGK1 GPA1-Gαs
7 7 Ass 2B pRET2 STE12
8 9 X3C pFIG1 ZsGreen
The table mentions the composition for each USER digestion reaction done for each of the samples in the above table.
SAMPLE QUANTITIES (μL)
10X USER enzyme 1
10X CutSmart Buffer 1
Dpn I 0.5
Promoter 1
Genes 1 μL of each (ex. receptors with linker)
Water 5.5
Total 10
Results

We obtained 8 different vectors each with a promoter and a gene as mentioned in the table above.

3. Making plates for E. coli to grow

Team members: Hitesh and Ojas

To make LB Agar + Carbenicillin medium in the petri dish to later plate the transformed colonies of E. coli.

Materials
  • LB Agar solidified medium
  • Carbenicillin Antibiotic
Procedure
  1. Heated the 250 mL LB-Agar solidified medium glass container in microwave for a min.
  2. Cooled down a bit and then in the lab bench, added 500 μL of Carbenicillin i.e a heat-resistant antibiotic that has the same activity as Ampicillin.
  3. Mix and transfer around 25 mL to each petri dish and made around 10 plates from the entire sample.
Results

Prepared 10 plates from 250 mL solidified LB Agar medium.

3. E.coli transformation

Team members: Hitesh and Ojas

To transform the USER reacted samples into E.coli

Materials
  • Competent E.coli cells
  • USER reacted samples
  • Ice box
  • LB media
Procedure
  1. The compentent E.coli thawed in the ice box
  2. 3 μl of each USER sample were added to their respective 50 μl competent cells containing tubes.
  3. 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.
  4. 1 ml of LB media was added to all the tubes, which were then incubated for 10 minutes.
  5. Centrifugation was carried out at 8000 g for 60 seconds and 900 μl of the supernatant was discarded.
  6. With the remaining 100 μl media, the cells were resuspended and then added on to the agar plates and were spread evenly.
  7. The plates named S1-S8 contain cells with USER samples from 1-8, respectively.
  8. There is a control plate (empty) without E.coli, just to check for contaminants.
  9. All the plates were finally sealed with parafilm and left inside the incubator to allow growth of colonies.
Data
SERIAL NUMBER VECTOR PROMOTER 1 μL GENE FRAGMENT 1 μL
1 X3A pCCW12 GPER
2 X3A pCCW12 XLHCGR
3 X3A pCCW12 GPER-linker + sfGFP
4 X3A pCCW12 XLHCGR-linker + sfGFP
5 Ass 2A pPGK1 GPA1-Gαi
6 Ass 2A pPGK1 GPA1-Gαs
7 Ass 2B pRET2 STE12
8 X3C pFIG1 ZsGreen


SERIAL NUMBER for plates VECTOR
1 X3A+pCCW12+GPER
2 X3A+pCCW12+XLHCGR
3 X3A+pCCW12+GPER-linker-sfGFP
4 X3A+pCCW12+XLHCGR-linker-sfGFP
5 Ass 2A+pPGK1+GPA1-Gαi
6 Ass 2A+pPGK1+GPA1-Gαs
7 Ass 2B+pRET2+STE12
8 X3C+pFIG1+ZsGreen
Results

After incubation at 37 C overnight, colonies were present in the following samples: 1, 2, 5, 6, 7, and 8. No colonies were seen in samples 3 and 4, and none were seen in the control plate. In sample 2, only 2 colonies were present. The experiment should be repeated for samples 3 and 4 as well as for sample 2. The fact that no colonies were seen in the control plate validates that the plates were not contaminated.

28th of May

1. colony PCR

Team members: Iben, Swenja & Ojas

To pick transformed E. coli colonies from the plate and do colony PCR. The colony PCR was done using the forward primer for the promoter and the reverse primer for the gene while the template was the vectors in the transformed E. coli colonies.

Materials
  • 10X X7 PCR buffer
  • X7 polymerase
  • F-primer
  • R-primer
  • dNTP
  • Template: Colonies from overnight plates
Procedure
  1. PCR tubes were prepared as per the normal PCR amplification protocol (Section 3, 22nd May 2019 ), however, the mix was scaled down so the final volume in each tube is 10 µl. See details in below Table 2.
  2. Primers were added to match the ligated fragments, i.e. forward primer for the promoter and reverse primer for the gene fragment as per below Table 3.
  3. Upto 7 PCR reactions were performed for each sample (S1, S2, S5, S6, S7, and S8) where possible. This should ensure to eliminate false positives. Samples 2 and 5 had only 2 and 6 colonies respectively, so for these samples only this number of reactions were prepared. Colonies were marked with numbers so we can return and collect material from positive colonies later on.
  4. A PCR plate was prepared, using the row number corresponding to the sample number.
  5. All components were added to the PCR mix, using a pipette tip to carefully collect material (template) from 7 different numbered colonies and transferring it to the corresponding PCR tube. X7 polymerase was kept in the freezer until we were ready to use it. It was thawed on ice, and added to the mix as the final component.
  6. The plate was spun to mix samples and remove bubbles before being placed in PCR machine.
  7. PCR was run for the same temperature and time as for normal PCR.
    1. PCR program: 30 cycles of:
    2. 98 degree celsius: 30 seconds
    3. 98 degree celsius: 20 seconds
    4. 55 degree celsius: 25 seconds
    5. 72 degree celsius: 2 minutes and 30 seconds
    6. 72 degree celsius: 5 minutes
    7. 12 degree celsius: Infinity
Data
Table 2: The composition of materials to be used per colony PCR amplification sample (NB: Reaction scaled down to 10 µl in each PCR tube)
Materials Quantity (μL)
10X X7 PCR buffer 1
dNTPs 0.8
F-primer (10 μM) 0.5
R-primer (10 μM) 0.5
Template ~ 0.1
X7-Polymerase 0.1
mQ Water 7


Table 3: Forward and reverse primers for each ligated gene construct
SAMPLE (Plate number) VECTOR (used to transfer E coli in that plate) F-PRIMER R-PRIMER
1 X3A+pCCW12+GPER pCCW12-F GPER-R
2 X3A+pCCW12+XLHCGR pCCW12-F XLHCGR-R
5 Ass 2A+pPGK1+GPA1-Gαi pPGK1-F GPA1-Gɑi-R
6 Ass 2A+pPGK1+GPA1-Gαs pPGK1-F GPA1-Gɑs-R
7 Ass 2B+pRET2+STE12 pRET2-F Ste12-R
8 X3C+pFIG1+ZsGreen pFIG1-F ZsGreen-R
The 96-well PCR plate arrangement with each column 1-8 corresponding to the samples (1-8) and the rows A to G corresponding to the colony number from the transformed E. coli plates.
1 (S1) 2 (S2) 3 (S3) 4 (S4) 5 (S5) 6 (S6) 7 (S7) 8 (S8) 9 10 11 12
A C1 C1 - - C1 C1 C1 C1
B C2 C2 - - C2 C2 C2 C2
C C3 - - - C3 C3 C3 C3
D C4 - - - C4 C4 C4 C4
E C5 - - - C5 C5 C5 C5
F C6 - - - C6 C6 C6 C6
G C7 - - - - C7 C7 C7
H
Results

We got the 6 PCR amplified product samples of the entire fragment with the promoter and the gene together for samples S1, S2, S5, S6, S7, and S8 that were then confirmed using gel electrophoresis.

29th of May

1. Gel electrophoresis

Team members: Swenja, Claudia and Ojas

To confirm the presence of promoter + gene fragments in the vector that was used to transform E. coli colonies. This helps confirm for positive transformed colonies.

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 in a new PCR tube with 1 μL of loading dye was added to each sample.
    3. To load the wells, the first and the last well were filled with the 1 kb DNA ladder while wells 2 to 13 were loaded with the colony PCR samples (S1-S8) and their respective colonies C1 and C2 while 14 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 (Plate number) VECTOR (used to transfor E coli in that plate)
1 X3A+pCCW12+GPER
2 X3A+pCCW12+XLHCGR
5 Ass 2A+pPGK1+GPA1-Gαi
6 Ass 2A+pPGK1+GPA1-Gαs
7 Ass 2B+pRET2+STE12
8 X3C+pFIG1+ZsGreen
Well 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Samples 1 Kb ladder S1C1 S1C2 S2C1 S2C2 S5C1 S5C2 S6C1 S6C2 S7C1 S7C2 S8C1 S8C2 1 Kb ladder

The sample numbers are labelled in reference to the colony PCR wherein the samples 1 to 8 are for each vector (includes promoter, gene and the backbone) and is referred to as S1 to S8 while for each sample was transformed into E coli. And grown in the plates to select for colonies which is abbreviated as C1 to C7 at max but gel electroporesis was done for maximum 2 samples only.

Results

For the sample S1, colony 2 was positive. For the sample S2, colony 1 was positive. For samples S5 and S7, both colonies 1 and 2 were positive. For samples S6 and S8, both colonies 1 and 2 were negative (Gel electrophoresis was done for colonies 3 to 8 to find positive ones) . Samples S3 and S4 did not have any colonies (To be done again).

2. Gel electrophoresis

Team members: Swenja, Claudia and Ojas

Gel electrophoresis was carried out for colonies 3 to 7 for samples 6 and 8 using the same protocol as used above because for these samples the colonies 1 and 2 did not give a positive result.

Data
Well 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Samples 1 Kb ladder S6C3 S6C4 S6C5 S6C6 S6C7 S8C3 S8C4 S8C5 S8C6 S8C7 1 Kb ladder
Results

For sample 6, colonies 4 and 5 were positive. For sample 8, colonies 4 and 5 were positive.

3. Transformed E.coli colonies into liquid cultures

Team members: Hitesh, Signe and Claudia

To inoculate the positively transformed colonies into liquid LB media and grow for vector amplification.

Materials
  • LB media
  • Carbenicillin (Antibiotic)
  • Falcon tubes
  • Inoculation loops
Procedure
  1. 200 μl of carbenicillin was added to the 100 ml LB media.
  2. 100 ml of the LB media was transferred into 10 falcon tubes, each containing about 7 ml. The tubes were named from 1 to 10.
  3. From the Gel electrophoresis results, one or more positive colonies were selected from each of the agar plates containing different samples.
  4. The transformed positive colonies were inoculated into the falcon tubes as in the table below
  5. The tubes containing the positive transformed colonies were kept in the shaking incubator (37 ℃) overnight.
  6. The plates were placed back in the fridge in the morning.
The table shows the information about the positive colonies picked from each plate. For e.g. Tube 1 contains colony 2 from the plate S1.
Revised Tube numbers SAMPLE (Plate number previously) Positive colonies VECTOR (used to transfor 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

30th of May

1. Plasmid DNA extraction

Team members: Benedicte, Mads & Anett

To extract plasmid DNA from the E. coli liquid cultures from 28th May.

Materials
  • E.Z.N.A. Plasmid DNA Mini Kit I.
Procedure
  1. We took out the tubes from the 37 °C incubator (ON from yesterday). There was growth in all of them.
  2. 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.
  3. We centrifuged at 10 000 g for 1 min (mistake: we did it at 12 °C instead of room temperature.
  4. We poured out the culture media and kept the pellet.
  5. We added 250 µl Sol I, vortexed the samples, transferred the suspension into new microcentrifuge tubes.
  6. We added 250 µl Sol II, inverted the samples and waited for 2-3 minutes (timing is important!)
  7. We added 350 µl Sol III, inverted the tubes.
  8. We centrifuged the samples at maximum speed for 10 minute. White pellet was formed.
  9. We skipped the optional column equilibration (because of the lack of some solutions).
  10. We transferred the supernatants into columns in 2 ml collection tubes.
  11. We centrifuged it for 1 min at maximum speed, discarded the filtrate.
  12. We skipped the 8. and 9. Step of the protocol (we did not have HBC solution a researcher recommend us to try it without).
  13. We added 700 µl DNA Wash Buffer, centrifuged it for 30d, discarded the filtrate.
  14. We repeated the previous step again.
  15. We centrifuged the empty column for 2 min at maximum speed to dry the column.
  16. We transferred the column into new microcentrifuge tubes.
  17. We added 50 µl Elution Buffer, let it sit for 1 minute and then we centrifuged it for 1 min at maximum speed.
  18. 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!
  19. 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
Results

After completing plasmid DNA extraction and purification using the Miniprep kit, we ran a gel and got the following result:

1 Kb ladder S-1 S-2 S-3 S-4 S-5 S-6 S-7 S-8 S-9 S-10 1 Kb ladder

From the left: ladder (1Kb Plus Ladder), sample 1 (GPER+pCCW12), sample 2 (XLHCGR+pCCW12), sample 3 (GPA1-Ga1+pPGK1), sample 4 (GPA1-Ga1+pPGK1), sample 5 (GPA1-Gas+pPGK1), sample 6 (GPA1-Gas+pPGK1), sample 7 (STE12+pRET2), sample 8 (STE12+pRET2), sample 9 (ZsGREEN+pFIG1), sample 10 (ZsGREEN+pFIG1), ladder (1Kb Plus ladder)

Expected sizes (theoretical sizes) Experimental sizes
1 Ca. 5Kb
2 Ca. 5Kb
3 Ca. 9Kb ?
4 Ca. 3Kb
5 Ca. 3Kb
6 Ca. 4Kb
7 Ca. 4Kb
8 Ca. 4Kb
9 Ca. 3.5Kb
10 Ca. 3.5Kb

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.

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