Team:UCopenhagen/Notebook/Week 31
Week 31 (29th of July-4th of August)
29th July 2019
1. GEL ELECTROPHORESIS OF E.COLI TRANSFORMATION CONSTRUCTS AND YEAST COLONY PCR SAMPLE
Team members: Swenja and Hitesh
Gel electrophoresis was performed for 8 constructs which have been previously (25th July) purified from E. coli transformation and the 4x eight colonies that resulted from the yeast transformation followed by colony PCR.
Materials
- 1% agarose
- 1x TAE buffer
- 1 kb DNA ladder
- Loading dye
- PCR tubes
- Templates
- Ice
Procedure
- To prepare the gel 1% agarose was taken from the common stock in electrophoresis room.
- The agarose-TAE buffer solution was poured into casting trays with 30 and 23 wells, respectively.
- The solidified gel was placed on the electrophoresis chamber filled with TAE buffer.
- To prepare the sample to be loaded and mixed. 4 μL of each sample were transferred into a new PCR tube and 1 μL of loading dye was added to each sample.
- The wells were loaded according to the images below. A negative control of the yeast colony PCR had been loaded into the last well of the second gel. A 1 kb DNA ladder was added before and 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.
Data
| Sample |
|---|
| 1. X3A-pCCW12-GPER |
| 2. X3A-pCCW12-GPER-sfGFP |
| 3. X3A-pCCW12-XLHCGR-sfGFP |
| 4. X3A-pCCW12-GPER |
| 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 |
Results
The results of the gels can be seen below. For the yeast transformation constructs 1 and 4, no positive colonies could be identified. Here, the constructs showed either no bands or bands at 1500 bp, meaning that after cutting at the restriction sites, no gene fragment or the wild type fragment had been inserted. For construct 2 of the yeast transformation, two colonies were positive as they showed a band at 1000 bp corresponding to X3C (2.4 and 2.7). Similarly, colony 3.3 was tested positive for the construct 3.
2. COLONY PCR OF TRANSFORMED E. COLI
Team members: Hitesh and Swenja
The E. coli transformed on 25th of July with Ass2A-pPGK1-GPA1-Gas was used to carry out colony PCR.
Materials
- 10X X7 Polymerase buffer
- X7 Polymerase
- F-primer for the promoter
- R-primer for the gene
- dNTP
- Template (Colonies from overnight plates)
- mQH2O
Procedure
- 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 table below.
- Primers were added to match the ligated fragments, i.e. forward primer for the promoter and reverse primer for the gene fragment.
- Ten PCR reactions were performed on ten colonies. This should ensure to eliminate false positives. The arrangement can be seen in the table below.
- For PCR, a master mix was prepared consisting of all materials but the templates and then 10 µl were added to eleven PCR tubes, respectively.
- With a pipette tip the material of each colony (template) was carefully collected and transferred to the corresponding PCR tube. To the last tube, 0.1 µl of water was added to account for a negative control.
- The tubes were spun down in the table centrifuge to mix the samples and remove bubbles before being placed in PCR machine.
- The standard PCR protocol was run.
Data
| 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 |
| Sample | F-primer | R-primer |
|---|---|---|
| pPGK1-GPA1-Gαs | pPGK1-F | GPA1-Gαs-R |
Results
Sample 4 ( in the fifth lane from the right) was not loaded. Otherwise, the wells were loaded with the following probes (from left to right (ladder, samples 1-10, negative control)).
3. COLONY PCR II OF TRANSFORMED YEAST FROM 22ND JULY 2019
Team members: Ojas
Colony PCR II was performed of the transformed yeast from 22nd July 2019.
Materials
- NaOH solution
- 2x MangoMix
- F-primer 1
- F-primer 2
- R-primer
- Template (colonies from overnight plates)
Procedure
- Ten more colonies (labelled 9-18) from the transformation (22nd of July) were picked from the plates with the 5-assembler system (two plates of GPER with Gαi) 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.
- The NaOH solution (hereafter referred to as dirtboil) with the yeast were then boiled for 10 mins at 99 °C.
- The master mix for the PCR reaction was prepared as per the table below. There was prepared enough mastermix for 21 reactions including a negative control (without template).
- The master mix was aliquoted into 33 PCR tubes after which 1 µl of dirtboil was added to each PCR tube (achieving a total volume of 10 µl).
- Hereafter, the PCR tubes were put in the thermocycler using the following program (b.-d. where run for 35 cycles):
- 96 °C for 30 s
- 96 °C for 20 s
- 55 °c for 25 s
- 72 °C for 90 s (1 min pr. kb)
- 72 °C for 5 min
- 12 °C indefinitely
- Thereafter, the samples were put into the -20 °C freezer.
Data
| Materials | Quantity (µl) |
|---|---|
| NaOH solution | 20 |
| 2x MangoMix | 5 |
| F-primer 1 | 0.5 |
| F-primer 2 | 0.5 |
| R-primer | 0.5 |
| mQ water | 2.5 |
| Total | 9 |
The composition of materials to be used per colony PCR amplification.
Results
Results missing
4. INOCULATION OF POSITIVE YEAST COLONIES OF CONSTRUCT X3A-pCCW12-GPER-sfGFP
Team members: Noel
The colonies of the plates that were transformed on the 22nd of July were inoculated into liquid Glu-U media.
Materials
- Glu-U media
- Colonies 4 and 7 from plate 2 (construct: X3A-pCCW12-GPER-sfGFP)
- Colony 3 from plate 3 (construct: X3A-pCCW12-XLHCGR-sfGFP)
Procedure
- The same protocol as the previous days was followed (protocol from July 5th), only exchanging the buffer from 10X X7 PCR buffer with 5X HF buffer. Since this buffer is only half as concentrated than the other one, twice of it and a respectively adjusted amount of water has to be used.
- The following primers were used:
- h-TPST1-F
- h-TPST1-R
- h-TPST2-F
- h-TPST2-R
- The following samples were PCR-amplified and loaded on the gel.
- TPST1 (1113 base pairs)
- TPST2 (1134 base pairs)
- Negative control (no template)
The samples of the colonies were taken, suspended into ca. 7 ml of Glu-U liquid media and grown O/N at 30° C.
30th July 2019
1. PLASMID PURIFICATION FOR Ass2A-pPGK1-GPA1-Gas EXPRESSING E. COLI
Team members: Noel and Hitesh
We had the culture from the 29th of July that expressed Ass2A-pPGK1-GPA1-Gαs. With this, we performed plasmid purification as described in the instructions of the EZNA Plasmid purification kit. The colony was split when transferred to the binding column with the collection tube and two preparations were performed. The resulting concentrations of the solutions were 69.7 and 61.7 ng/µl.
2. FLUORESCENCE MICROSCOPE OF POSITIVE YEAST COLONIES
Team members: Noel and Hitesh
Samples of the same yeast cultures (Plate 2, colony 4 and 7; Plate 3, colony 3) that were inoculated the day before were analysed under the fluorescence microscope.
Results
As seen in the pictures, both chosen yeast colonies of plate 2 express the GFP-fused receptor and show therefore a green fluorescence light. The colony of plate 3 doesn’t show green fluorescence which was expected as a frame-shift mutation was identified in the sequence.
31st July 2019
1. PCR AMPLIFICATION of TPST1 & TPST2
Team members: Iben and Ojas
Procedure
Data
| Materials | Quantity (µl) |
|---|---|
| 5X High Fidelity PCR buffer | 10 |
| dNTPs | 4 |
| F-primer (10 μM) | 2.5 |
| R-primer (10 μM) | 2.5 |
| Template | 0.5 |
| X7-Polymerase | 0.5 |
| mQ water | 30 |
| Total | 50 |
Results
The first gel we ran contained TPST1 in the first well, TPST2 in the second, and negative control in the third. The band for TPST2 was positive and corresponds well to the expected 1134 bp, as it lies between the 1000 and 1650 base pairs bands in the ladder. However, the TPST1 sample was negative. It is possible that we forgot to add template in the first sample, therefore we redid the experiment with TPST1.
In this second experiment, TPST1 (well 1) is also positive, and corresponds well to the expected 1113 base pairs. The next step will be USER ligation of the fragments.
1st August 2019
1. YEAST TRANSFORMATION
Team members: Iben and Jonas
Yeast transformation was performed according to the protocol from the 22nd of July.
Procedure
The following 4 strains were transformed:
| Sample |
|---|
| 1. Ass2C+GPER+Gαi+STE12+ZsGreen |
| 2. Ass2C+GPER+Gαs+STE12+ZsGreen |
| 3. Ass2C+XLHCGR+Gαi+STE12+ZsGreen |
| 4. B(Ass2)+XLHCGR-sfGFP+X3C |
The concentration, volume and amount of DNA was calculated for all the DNA samples used:
| Sample | Stock conc. (ng/µl) | Amount needed |
|---|---|---|
| X3A-pCCW12-GPER (1000 ng) | 108.5 ng/µ | 1000/108.5 = 9.22 µl |
| Ass2A-pPGK1-Gαs (1500 ng) | 94.4 ng/µl | 15.89 µl |
| Ass2A-pPGK1-Gαi (1500 ng) | 118.8 ng/µl | 12.63 µl |
| Ass2B-pRET2-STE12 (1500 ng) | 114.05 ng/µl | 13.15 µl |
| Ass2C (1500 ng) | 219.7 ng/µl | 6.83 µl |
| X3C-pFIG1-ZsGreen (1500 ng) | 122.05 ng/µl | 12.29 µl |
| X3A-pCCW12-XLHCGR (1000 ng) | 203.7 ng/µl | 4.91 µl |
| X3A-pCCW12-XLHCGR-sfGFP (600 ng) | 144.7 ng/µl | 4.15 µl |
| BAss2 (900 ng) | 287.5 ng/µl | 3.13 µl |
| X3C (900 ng) | 286.8 ng/µl | 3.14 µl |
Data
| Sample | X3A module | µl | Ass2A module | µl | Ass2B module | µl | Ass2C module | µl | X3C module | µl | Total (µl) |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | X3A-pCCW12-GPER | 9.22 | Ass2A-pPGK1-Gαi | 12.63 | Ass2B-pRET2-STE12 | 13.15 | Ass2C | 6.83 | X3C-pFIG1-ZsGreen | 12.29 | 56.1 |
| 2 | X3A-pCCW12-GPER | 9.22 | Ass2A-pPGK1-Gαs | 15.89 | Ass2B-pRET2-STE12 | 13.15 | Ass2C | 6.83 | X3C-pFIG1-ZsGreen | 12.29 | 56.1 |
| 3 | X3A-pCCW12-XLHCGR | 4.91 | Ass2A-pPGK1-Gαi | 12.63 | Ass2B-pRET2-STE12 | 13.15 | Ass2C | 6.83 | X3C-pFIG1-ZsGreen | 12.29 | 56.1 |
| 4 | X3A-pCCW12-XLHCGR-sfGFP | 4.15 | BAss2 | 3.13 | X3C | 3.14 | 11.08 |
| Sample | Total volume (µl) | Volume of buffer (µl) | Volume of enzyme (Not1) (µl) |
|---|---|---|---|
| 1 | 58.38 | 6.49 | 1 |
| 2 | 55.12 | 6.12 | 1 |
| 3 | 54.07 | 6.69 | 1 |
| 4 | 11.42 | 1.27 | 1 |
Results
Results missing
2nd of August
1. Inoculation of O/N culture for competent cell preparation
Team members: Iben
1 culture from seed E. coli plate was inoculated in 10 mL LB and grown O/N at 37 degrees.
4th August 2019
1. E.coli competent cell test
Team members: Iben and Noel
Materials & Procedure
The iGEM competent cell kit was used to test the competency of our E.coli cells. Therefor, the standard protocol was used.
Results
The plates showed the wished-for red color revealing a successful transformation of our competent cells.
Left: the 10 pg/µl transformation. Right: The 100 pg/µl transformation.
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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iGEM_Copenhagen
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