Team:UCopenhagen/Notebook/Week 23







Week 23 (3rd-9th of June)

4th of June

1. Sequencing

Team members: Swenja & Claudia
Sequencing of the vector DNAs.

Materials
  • Primers
  • DNA
  • mH20
Procedure
  1. The concentration of each purified plasmid DNA sample was measured using NanoDrop.
  2. A stock solution of the primers was prepared by adding 10 µl of each primer to a new eppendorf tube containing 90 µl mH20, respectively. The stock solutions were stored in the freezer after usage.
  3. 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.
  4. 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 volume of 10 µl
  5. Stickers with QR codes have been given to us by Nat. 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.
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
The table shows nanodrop readings for each sample
Construct Sample Nr. A60/280 A260/230 ng/µl µl used
GPER + pCCW12 1 1,87 1,86 - 2,33 2,34 - 126,4 131,3 - 3,9
XLHCGR + pCCW12 2 1,78 1,82 1,81 1,61 1,85 1,85 194,3 151,1 151,9 3,3
GPAI-G𝛼i + pPGK1 3 1,85 1,88 - 2,32 2,35 - 94,1 94,6 - -
3 C4 - - - - - - 66,5 63,9 - 8,3
3 C5 - - - - - - 60 - - 7,7
4 1,91 1,86 - 2,48 2,45 - 79,6 80,9 - 6,2
GPAI-G𝛼s + pPGK1 5 1,90 1,84 1,87 2,36 2,37 2,32 67,1 93,3 83,6 6,1
6 1,89 1,87 - 2,41 2,37 - 55,2 56,9 - -
STE1 + pRET2 7 1,88 1,86 1,85 2,32 2,25 2,22 123,4 80,4 78,5 6,3
8 1,91 1,92 - 2,39 2,39 - 76,6 68,7 - -
ZsGreen + pFIG1 9 1,96 1,87 - 2,40 2,35 - 66,1 68,0 - -
10 1,83 1,83 1,86 2,29 2,34 2,34 150,1 107,6 111,3 4,1
Construct Sample Nr. 1. Primer 2. Primer 3. Primer
GPER + pCCW12 1 YEA74 YEA81 GPER-F
XLHCGR + pCCW12 2 YEA74 YEA81 XLHCGR-F
GPAI-G𝛼i + pPGK1 4 YEA75 YEA137 GPAI-F
GPAI-G𝛼s + pPGK1 5 YEA75 YEA137 GPAI-F
STE1 + pRET2 7 YEA138 YEA139 STE12-F
ZsGreen + pFIG1 10 YEA137 YEA84 ZsGreen-F

Results

Construct Primers+Sequencing label Analysis
X3A-pCCW12-GPER

YEA74+1CB8ZAB425

YEA81+1CB8ZAB426

GPER-F+1CB8ZAB427

 There is a missing base in 6420, causing a frameshift.
X3A-PCCW12-XLHCGR

XLHCGR+1CB8ZAB428

YEA81+1CB8ZAB429

YEA74+1CB8ZAB430

 Missing bp at 1154 causing frameshift.
GPAI-G𝛼i + pPGK1

GPA1+1CB8ZAB431

YEA137+1CB8ZAB432

YEA75+1CB8ZAB433

 Perfect.
GPAI-G𝛼s + pPGK1

GPA1+1CB8ZAB434

YEA137+1CB8ZAB435

YEA75+1CB8ZAB436

 Frameshift from 2795
STE1 + pRET2

STE12+1CB8ZAB437

YEA138+1CB8ZAB438

YEA139+1CB8ZAB439

 We were unable to align this Sequence.
ZsGreen + pFIG1

YEA84+1CB8ZAB440

ZS-GREEN+1CB8ZAB441

YEA77+1CB8ZAB442

 Silent mutation in base 729. Otherwise correct sequence. Template was only the USER zsGREEn - without vector and promoter.

5th of June

1. USER-Ligaiton/E.coli transformation part 2: "Sample 3 & 4"

Team members: Hitesh and Noel

Repeating 10. Adapted USER-Ligation and Transformation of Sample 3 and 4 as we didn’t get any colonies when E coli. was transformed with Sample 3: pCCW12-GPER-linker-sfGFP and Sample 4: pCCW12-XLHCGR-linker-sfGFP

Materials
  • Sample 3: pCCW12-GPER-linker-sfGFP
  • Sample 4: pCCW12-XLHCGR-linker-sfGFP

Per sample:

  • 10x User enzyme 1µl
  • 10x cut smart buffer 1 µl
  • Dpn1 0.5 µl
  • Milli-Q 5.5 µl
  • X3A vector 1 µl
Procedure
  1. Unify the USER enzyme, the cut smart buffer, 1 µl of promoter, 1 µl of receptor, 1 µl of sfGFP and 5.5 µl of mili-Q-water in a PCR tube
  2. Let the samples ligate in the PCR machine using the following program: 37° C for 30 min->80°C for 20 min, 25°C for at least 15 minutes
  3. Add 1 µl of the corresponding vector which was X3A in this case for both the samples
  4. Put it back into the PCR machine: 37° C for 10 min->25° C for 10 min-> 20° C for 10 min-> 12 °C for infinity
  5. For the transformation the protocol that is found under “15” 29th May 2019 was used, yet the following changes were implemented:
    • 5 instead of 3 µl were added to 50 µl of competent cells.
    • After adding the media to the cells a 60 minutes recover period 37 °C was given to the cells
Results

It worked for sample 3 as we got colonies, but sample 4 was still not compliant. More colonies compared to the first E.coli plates were seen.

6th of June

1. Colony PCR for sample 3 and 4

Team members: Iben & Jonas; Ojas and Benedicte

E coli transformed with Sample 3: pCCW12-GPER-linker-sfGFP and Sample 4: pCCW12-XLHCGR-linker-sfGFP were in LB agar plates grown overnight which were collected. We picked transformed E. coli colonies from the plate and did 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 Polymerase buffer
  • X7 Polymerase
  • F-primer for the promoter
  • R-primer for the gene
  • dNTP
  • Template (Colonies from overnight plates)
  • mH2O
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. 6 PCR reactions were performed on 6 colonies for each sample (S3 and S4). This should ensure to eliminate false positives. The arrangement can be seen in the table below.
  4. For PCR, a 96 well plate was prepared, using the column number as the sample number (that is the plate that has transformed E coli colony with either Sample 3: GPER linker + pCCW12 + sfGFP and Sample 4: XLHCGR + pCCW12 + sfGFP) while the row number corresponding to the colony number picked from the respective plate.
  5. All components were added to the PCR mix, using a pipette tip to carefully collect material (template) from 6 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 place 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.
  8. After first run on the gel electrophoresis, it was confirmed that we got no positively transformed colony for sample:4 and therefore the colony PCR was repeated for all the 14 colonies of sample S4 again.
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
Forward and reverse primers for each ligated gene construct
SAMPLE F-PRIMER R-PRIMER
S3 PCCW12-F sfGFP-R
S4 PCCW12-F sfGFP-R
The 96-well PCR plate arrangement with column 1 and 2 corresponding to the samples (3 and 4) and the rows A to F corresponding to the colony number from the transformed E. coli plates.
1 (S3) 2 (S4)
A C1 C1
B C2 C2
C C3 C3
D C4 C4
E C5 C5
F C6 C6
The 2 sets of PCR tubes with 14 colonies of E coli. Transformed with Sample 4 as arranged for colony PCR.
S4C1 S4C2 S4C3 S4C4 S4C5 S4C6 S4C7
S4C8 S4C9 S4C10 S4C11 S4C12 S4C13 S4C14
Results

We got the 6 PCR amplified product samples of the entire fragment with the promoter and the gene together for samples S3 and S4. After first gel electrophoresis it was found, that the 6 colonies of E coli. transformed with Sample 4 did not yield positive transformation and therefore colony PCR was repeated for all the 14 colonies for the E coli. Transformed with Sample 4.

2. Gel electrophoresis

Team members: Benedicte and Ojas (Benedicte and Anett on the 7th)

To confirm the presence of promoter + gene fragments in the vector that was used to transform E. coli colonies. To do so, we performed colony PCR with primer such that it shall amplify the promoter + gene fragment from the plasmid and then we run gel that helps confirm for positive transformed colonies. The first gel electrophoresis was done on 6 colony PCR amplified samples from each plate that had E. coli colonies. It was found that all the 6 colony PCR samples for the sample S4 failed to give a distinct band. Therefore, we repeated the colony PCR for Sample 4 again with all the 14 colonies on the plate. This set of 14 amplified samples for Sample S4 were again run on gel electrophoresis.

Materials
  • Agarose
  • 1X TAE-buffer
  • Loading dye
  • DNA ladder
  • PCR tubes
  • Ice box
Procedure
  1. To prepare the gel
    • 1% agarose gel was taken from the common stock in electrophoresis room
    • 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
    • Solidified gel was placed on the electrophoresis chamber filled with TAE buffer
    • 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.
    • 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 (colonies C1-C6) for each respective sample S3 and S4 while 14 was loaded with negative control.
    • Next, the gel electrophoresis was repeated for the 14 colony PCR amplified samples from the plate of E coli transformed with Sample S4.
  3. Finally the electrophoresis setup was put to 100 V in the electric chamber and was run for 25 mins.
Data
SAMPLE Plasmid construct F-PRIMER R-PRIMER
S3 pCCW12-GPER- linker-sfGFP pCCW12-F sfGFP-R
S4 pCCW12-XLHCGR-linker-sfGFP pCCW12-F sfGFP-R
well 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Gel 1: Samples 1 Kb S3C1 S3C2 S3C3 S3C4 S3C5 S3C6 S4C1 S4C2 S4C3 S4C4 S4C5 S4C6 1 Kb
Gel 2: Samples 1 Kb S4C1 S4C2 S4C3 S4C4 S4C5 S4C6 S4C7 S4C8 S4C9 S4C10 S4C11 S4C12 S4C13 S4C14 1 Kb
Results
For sample S3 colony 4 and 5 (C4 and C5) were positive. These colonies were grown overnight in liquid media as per 29th May 2019.
None of the sample S4 colonies were positive (Gel 1), so we completed another colony PCR with all the available sample 4 colonies (which were a total of 14 colonies) as described above, and we were still unable to get a positive with any of them (Gel 2)

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