Notebook
Here you can follow our progress throughout the summer and fall.
Team:Linkoping Sweden/Notebook
Week 44
With our transformed bacteria fluorescence was measured after induction with arabinose in different concentrations. The result from the sequencing showed a 99% match. The part was then submitted to the registry (BBa_K2474000).
Week 43
A new substrate was assembled, our Amyloid beta-mNeonGreen fusion protein with the arabinose promoter p(bad). The substrate was transformed into BL21 bacteria. A plasmid prep was done and the part was sent to sequencing.
Week 42
Fluorescence of GFP in the transformed biobrick, and optical density was measured after induction to characterize the pbad promotor in biobrick K75000.
Week 41
We sent our substrate to sequencing. Meanwhile a biobrick, K75000, was dissolved and electroporated into bacteria for further characterization.
Week 40
The bacteria with egfp-tau were induced with different concentrations of arabinose under different temperatures. The measurement of optical density and the fluorescence showed similar results between the samples. We sent our substrate to sequencing.
Week 39
The egfp-tau substrate is in the vector pcb1A3 and for submission of our biobrick we needed to change it to a psb1C3 vector. The substrate and psb1c3 were cut and ligated.
Week 38
Continued test on the assembly of our chaperone system. This time we first tried to assemble groEL\ES and DnaK without the vector. groEL\ES and the vector were then cut by restriction enzymes and ligated together. The product was transformed into bacteria but no growth was found the next morning.
Week 37
This week we tried to assemble the superduper plasmid. GroEL/ES, TF and Dnak were dissolved and assembled with the vector psb1C3. Unfortunately the gel after a pcr of the assembly product revealed no assembly of the superduper plasmid.
Week 35
We had a possible successful assembly of our egfp-tau substrate. several assembly products were transformed. After the colony screening one had the right band size. A plasmid preparation was made from the colony.
Week 34
Continued ligation and assembly trials, trying different parameters. We also added purification steps after and between each method. Troubleshooting of the ligation and the earlier assembly experiments.
Week 33
With the pbad primer we amplificated pbad with a vector overhang. With this overhang we unsuccessfully tried to ligate pbad with the vector. We tried different restrictions sites and ligation methods like blunt end ligation and preparation with alkaline phosphatase. The results were bad as usual.
Week 32
Our Model has been finished now! After several weeks of hard work it has been completed and now is ready for consulting the protein expression. We phave now received a pBAD-primer so we can try to clone pBAD and pSB1A3 together.
Week 31
We practiced our standard experiment with cells containing premade construct. Cells with the tau-egfp and AB-egfp together with different chaperones were induced and fluorescence was measured after different inducing times.
Week 30
A new assembly with pad, EGFP and vector was started. This time we tried different reaction temperatures during the assembly reaction. We also tried to assemble pad mNeongreen and the vector. Both assemblies were unsuccessful.
Week 29
This week has been a week of troubleshooting. We have now added several controlling gels to our standard procedure and have almost pinpointed the problem. Hopefully we now will be able to fix this and continue with our planned experiments next week!
Week 28
The DNA has arrived! After some thorough calculation we started to assembly a plasmid with PSB1A3, pBAD and EGFP. Sadly thou, we failed and could not see any EGFP after heat-shocking and inducing. Next week we’ll troubleshoot and try again!
Week 27
Our DNA-fragments are being made over at IDT and we are mainly waiting. While we are waiting, we are cloning up the iGEM vectors PSB1C3 and PSB1A3 which we will be using as backbones in our plasmids later on.
Week 26
The DNA-inserts are now ordered. A total of 10 fragments have been ordered and will be assembled in different combinations to form 5 different plasmid all including an iGEM-backbone. We are also continuing testing our upcoming labs to give ourselves experience
Week 25
This week have mainly consisted of constructing our plasmids. We are now almost done with both the plasmid for the chaperones and the fusion proteins. Now we have to decide what inserts we are going to order and to make them compatible with our assembly later.
Week 24
Summer has now begun and finally we are ready to spend our full focus on iGEM! A provisory wiki has been published and in the laboratory we are training and testing the main labs we are going to use during the summer. We have also begun constructing our plasmids for the chaperones to be ordered from IDT.
