Team:SDU-Denmark/Notebook

Lab Journal

Week 25 (16/6 - 22/6)

Monday 16/6

The lab crash course began. We talked a lot about the project goal and the way of getting there. The team is now on the same page and ready for a great summer of exciting and interesting work to begin. In the lab we learned where everything was located and learned how to run a PCR.

Tuesday 17/6

In the lab: The PCR from yeasterday didn't work, so they were repeated. This time 50 µL PCR reaction were prepared. Gel purification was performed on the PCR products and the final concentration was measured by nanodrop.

Wednesday 18/6

The XbaI enzyme didn't work to cut plasmid 161 tuesday and we wanted to find out what was wrong. We had different theories as to what went wrong tuesday:

• Maybe the XbaI didn't have enough time to cut the plasmid. We only gave it 5 minutes.
• Maybe the restriction site on plasmid 161 didn't work.

1. We wanted to do the same as on tuesday, but allow the enzyme to work for 15 minutes instead of 5 minutes, in case this was our mistake.
2. We wanted to use the same plasmid as tuesday but cut it with EcoRI to see if this restriction site worked.
3. We wanted to try to cut a new plasmid (RFP) with the same enzyme for the same amount of time (5 minutes), to find out if the enzyme worked or not.

We also recieved lab safety training

Thursday 19/6

To be written.

Friday 20/6

Team page and Notebook page added to the wiki.

We tried to construct the TetR without the LVA-tag, 3 PCRs were made.

Saturday 21/6

All the pipettes were calibrated

A K12 MG1655 strain was plated out on agar, to colonize overnight, in order for us to produce more plasmid later on.

Sunday 22/6

A coloni from the overnight plate was transferred to a bulb with 10 µL LB and TSB buffer was prepared using the SOB.

1,5 µL plasmid (65,3 ng/µL) was added and 1 hour at 37*C were given for fenotypical expression. Everything else was done according to the SOP. The transformation was put on a chloramphenicol agar plate and left overnight in the heating cabinet.

Week 26 (23/6 - 29/6)

Monday 23/6

The transformation from yesterday did not work.

Agar plates with antibiotics were created. Ampicillin (50µg/mL), Kanamycin (25µg/mL) and Chloramphenicol (33µg/mL).

Wednesday 25/6

Transformation is attempted again today.

The e. Coli coloni was taken from the agarplate made on Saturday 21/6

Transformation was attempted with the four different plasmids: aza (from Ann Zahle), pSB1C3, pSB1A3, pSB1K3 (taken from igem kit 2013).

The transformed E. coli with the different plasmids, are spread onto agar plates with antibiotics (chloramphenicol, chloramphenicol, ampicillin, kanamycin respectively), and grown overnight.

Tomorrow the agar plates should be examined for any colonies.

- Jens J and Anne K

Thursday 26/6

The results from yesterday's transformation showed that the E. coli culture with the plasmid aza (from Ann Zahle) had grown on the agar plate with ampicillin. The transformation of the E. coli culture with pSB1A3 was also successful. The transformation of the E. coli with pSB1C3 and pSB1K3 respectively were not successful, and should therefor be repeated.

A colony from each of the successful cultures were prepared for overnight growth in LB medium with ampicillin. Additionally an overnight culture of the wild type E. coli strain without the plasmid was prepared.

The cultures should be prepared for storage at -80°C tomorrow.

- Jens J

Friday 27/6

The overnight cultures from yesterday were prepared for storage at -80°C by mixing a sample from each culture with glycerol. The samples were then stored at -80°C.

- Jens J and Camilla

Saturday 28/6

A new transformation was attempted with the plasmids pSB1C3 and pSB1K3. Two transformations were done of each plasmid. The cultures were put on agar plates with chloramphenicol and kanamycin respectively. Tomorrow the plates should be examined for cultures.

- Jens J and Camilla

Sunday 29/6

The transformation was partially successful. There were some, but very few, visible colonies on the agar plates. Two overnight cultures were prepared from one colony respectively of the strain with kanamycin and chloramphenicol resistance.

- Jens J and Camilla

Week 27 (30/6 - 6/7)

Monday 30/6

The overnight culture was not red, which means that something was wrong with the plasmid. Therefor a new overnight culture was prepared, so that colony pcr can be performed on the cultures. Furthermore an overnight culture was prepared of the wild type E. coli strain, so that it can be used for transformation tomorrow.

- Jens J

Tuesday 1/7

Today colony PCR (iGEM2014_SOP0021) was performed on the E. coli MG1655 strains with the plasmids: pSB1A3, pSB1C3, pSB1K3 and J04450 (also known as aza). The results showed that the constructs all had approximately the same weight, just below 1500 bp. This corresponds to the theoretical weight of the constructs, which is 1382 bp.

The development of the construct used to produce lacI was commenced. This was done by first running Phusion PCR (igem2014_SOP010) on lacI (BBa_I732100, Template 8A, Plate 3). The PCR product was then analysed on gel-electrophoresis and purified.

Transformation was performed on the plasmids pSB1C3 and pSB1K3 as the former transformation was unsuccessful. Furthermore transformation was also performed on the parts BBa_R0010 (lacP promoter) and BBa_E0840 (GFP coding seq.). The transformed E. coli were put on agar plates with antibiotic according to their respective resistance. The plates were placed in the heating cabinet and should be examined for colonies tomorrow.

-JJ

Wednesday 2/7

Because the PCR on lacI from yesterday yielded a low concentration, Phusion PCR was repeated today, where both the PCR product from yesterday and BBa_I732100 from iGEM were used as template. The results showed that the PCR product from yesterday was too short and still yielded a low concentration. While the PCR reaction with BBa_I732100 as template produced a higher concentration of lacI.

The transformation on the parts BBa_R0010 (lacP promoter) and BBa_E0840 (GFP coding seq.) from yesterday was unsuccesful so the experiment was repeated today.

Freezing stocks of E. coli with pSB1C3 and pSB1K3 were stored at -80°C. Each culture were mixed with 50% glycerol

Furthermore fast digest on our 3 PCR products were made with XbaI and SpeI

PCR 1 and 3 were ligated and PCR 2 and 3 were ligated. they are stored at 16°C overnight.

Thursday 3/7

Today the ligated PCR1/3 and PCR2/3 was purified by running them through a gel. The concentration of the purified constructs were measured by nano drop:

In order to create "sticky ends" on PCR1/3 and PCR2/3, the parts were fast digested. Also pSB1C3 was fast digested, so that it could be used as a backbone. The different fast digested parts were gel purified. PCR1/3 was then ligated to the backbone (pSB1C3) and PCR2/3 was ligated to the backbone (pSB1C3).

Colony PCR was performed on a colony from the yesterday transformation of the lacP promoter (BBa_R0010) and GFP coding seq. (BBa_E0840). Because the results showed that the sequences consisted of more base pairs than they theoretically should, a new colony PCR was performed on a different colony.