Team:Linkoping Sweden/Notebook

Week 25

2015-06-15 - 2015-06-21

Biobricks were resuspended and transformated. The transformation didn’t give any good results. Non-transformed bacteria was tested on plates and in falcon tubes without antibiotics, the bacteria grew. The bacteria with antibiotics in a falcon tube didn’t grew. This indicated that there wasn’t anything wrong with the bacteria, plates, SOB or the antibiotic. A new transformation was made with a refined protocol and with a x1 dilution which gave better results. The colonies were restreaked and cultivated in falcon tubes. Later on a plasmid preparation was made but the concentration was quite low.

Week 28

2015-07-06 - 2015-07-12

Heatcompetent cells were made and used when transforming the biobricks. The few colonies visible were centrifuged down and restreaked and left over night. The next day they were left in falcon tubes to grow for another night. A plasmid preperation was made, the result was mesured with nanodrop and gave more stable concentrations than earlier, even though some of them were still quite low.

Name Conc. ng/µl 260/280 260/230
J4650 (1) 60.0 1.90 2.43
J4650 (2) 25.9 1.94 2.84
K1319002 (1) 48.4 1.89 2.49
K1319002 (2) 48.7 1.87 2.53
K525998 (1) 21.6 1.89 3.62
K525998 (2) 20.9 1.88 2.49
E1010 (1) 41.9 1.87 2.52
E1010 (2) 26.0 1.91 3.38
J04600 (1) 23.5 1.83 2.85
J04600 (2) 25.0 1.81 2.30

Week 29

2015-07-13 - 2015-07-19

A transformation on the biobrick E1010, attatched to pSB1C3, was made. Bacteria had grown on the control and the other plates were overgrown. A restreak was still made. The results looked good and the control was empty. An over night culture was made in falcon tubes.

Week 30

2015-07-20 - 2015-07-26

After two plasmid preperations on E1010 the concentration was stable around 30 ng/µl. In case the quite low concentrations were due to a low growth in the over night cultures a new over night was made. Several colonies were picked from the restreak to increase the yeild. Digset, ligation and screening was made on the biobricks and the psb1C3 backbone, unfortunately the molecules wandered out of the gel. The problem was probably a too wigh voltage. A transformation was still made on some of the plasmids. A new digest and ligation was made, this time we cleaned our plasmids between the digest and ligation. The concentrations were too low to continue with a transfomation. Parallel a second digest and ligation was made and followed up with a screening.

Week 32

2015-08-03 - 2015-08-09

The week started with digestion of epitope 2, which was later controlled on an agarose gel. Later this week an efficiency test was conducted on our different digestion enzymes (Pst1. Xba1 and EcoRi).

Amplification of psb1c3 was conducted via PCR.

Towards the end of the week attempts were made to ligate epitope 2 and k525998 on pSB1C3. The ligate were checked on an agarose gel and then transformed into XL1 heat competent cells.

Week 33

2015-08-10 - 2015-08-16

The transformed ligate from the previous week was screened (first PCR then an agarose gel) and cultivated over night. The amplified psb1c3 was screened in the same manner. A plasmid preparation was conducted on the over night culture.

Epitope 2+ k525998 plasmids on pSB1C3 was then sent to sequencing. At the same time was the epitope 2 + k535998 plasmid digested and ligated with digests from E1010, K1319002 and J04500 respectively. We also conducted digestion and ligation on epitope 2 and J04650. Transformation was conducted on all ligations.

Over night and screening was then conducted on the transformed colonies. An efficiency test was conducted on newly made heat competent cells.

Plasmid preparations was made on the over night cultures.

Week 35

2015-08-24 - 2015-08-30

When recieving the results from the sequencing we realised that only one of our constructs were successful; K525998+epitope 2+ E1010. We continued to work with only this construct.

Next we conducted mutagenesis with the aim to remove a stopcodon that we discovered in our coding sequence, this proved to be difficult and several attempts were made. Finally we think we succeeded and made reastreaks on the colonies.

At the same time we continued working on the protein expression of RFP.

Week 26

2015-06-22 - 2015-06-28

New falcon tube-preparations were made from previous restreaks. A plasmid preparation was made and the concentration was measured. Some plasmids gave acceptable results but there was still room for aprovement.

Week 27

2015-06-29 - 2015-07-05

New colonies from previous transformations were prepared in falcon tubes to be cultivated over night. A plasmidprep was made and measured with nanodrop, the results were lower than before. Several new falcon tube-cultivations were made and incubated. The nanodrop showed a great variation in concentration. A screening was made, mostly for practice. The result was quite messy but educational.

Well nr. Name bp biobrick pb + PSB1C3 bp
2, 12 GeneRuler see below
3, 5 E1010 706 2776
7, 9 K131900 711 2781
11, 13 J04500 220 2290
15, 17 J04650 843 2913
18, 20 K525998 32 2102

An attempt on making heatcompetent cells were made and followed up with an efficiency test with poor results. The efficiency was between `1 * 10^6` and `3 * 10^7` cfu/μg (clu = colony-forming unit). A new attempt was prepared for the following week.

Week 31

2015-07-27 - 2015-08-02

A new digest was made on the backbone puc57 and our synthesized epitope. This was later put on an agarose gel. We could not see any digested plasmids. New digests on the backbone and epitope were made. This time the separation was visible on the gel.

Week 34

2015-08-17 - 2015-08-23

Starting off this week we continued with the epitope 2 + J04650 plasmid and ran a digest/ligation on it with E1010, K1319002 and J04500 respectively and afterwards a transformation was made for each ligation. Preparations were also made for the making of electrocompetent cells. We then made over night cultures from the transformations.

We received the sequencing results from the first ligations showing success in 3 of the 5 samples we sent. Plasmid preparations was made from the over night cultures followed by a screening of the plasmids.

Electrocompetent was made for the use of protein expression later on.

GFP and RFP was transformed into BL21 Gold cells. The GFP transformation was unsuccessful, a reastreak was made from the RFP. Furthermore, over night cultures was made from RFP.