Tested the transformation efficiencies of the NEB 10-beta and BL21(DE3) cells.
First designs for DNA-bridges and GIGA-BLOCKS (gBLOCKs) were made in SnapGene.
Transformed the BioBricks from the iGEM distribution kit 2019 into D5alpha competent cells. With the transformation protocol of iGEM.
Isolated the DNA of the BioBricks with a miniprep kit from Macherey Nachel.
Digested the Biobricks and and put in on a 0,8% agarose gel to visualize wether the enzymes can actually cut the biobrick out of its vector.
pSB1A3 and pSB1C3 were transformed and than isolated with miniprep kits from Zymor Research. Unfortunally the yield was not that good to work with because we stored a certain buffer not at 4 degrees Celsius.
Week 2:
Isolated pSB1A3 and pSB1C3 again with a miniprep kit.The yield here was again not that good. We think that the quality of the buffer was not good anymore after our storage mistake.
Our product designer came with the first concepts of our HRDK product.
Week 3:
Isolated pSB1A3, pSB1K3 and pSB1C3 again with a maxiprep kit.
Digested the maxipreps and put it on a 0,8% agarose gel.
Made Rosetta competent cells and tested the transformation efficiency.
Recieved TEV-cleaving site and bla fragments.
Product designer performed experimental tests to see how biodegradable the product really is.
Week 4:
Protein induction and purification of TEV, BRCA2-F6 and HSF2BP.
Characterisation of the TEV and BRCA2-F6 purification to see whether the protein was purified or not by SDS-PAGE analysis. It did not work.
Consultation with Erasmus MC Research Centre to bring sponsorship on track to see whether we can get BRCA2-F6 and TEV protein samples as a gift to our research.
July
Week 1:
Ordered the DNA Bridges at Twist Bioscience.
Protein induction and purification of TEV, BRCA2-F6 and HSF2BP again.
We tested wether the purification actually contains our product of interest, but it didn't. HSF2BP was successfully purified with our AKTA-system and was seen on SDS-PAGE gel.
Erasmus sponsorship rounded up. We got BRCA2-F6 and TEV samples to work with.
We did a PCR of the TEV-cleaving site and bla fragments.
Purificated the fragments and performed a fusion PCR to fuse the two fragments to each other.
Week 2:
Digested the PCR fusion product at the end of the DNA to make it possible to ligate it into pSB1C3.
The ligation product was transformed into DH5alpha cells.
A miniprep of the transformants was made and undergone a digestion check to see whether the construct really is present or not.
We had students come by from a high school and gave them an introduction in the lab and gave them multiple tasks to work on in the lab, to give them real-life lab experience.
Ordered the GIGA-BLOCKs (gBLOCKs) with our fusion proteins at IDT.
Week 3:
Digested again the PCR fusion product and pSB1C3, because the digestion check did not give good results.
The digest was transformed into DH5alpha cells.
August
Week 1:
Transformation of the fusion PCR product with pSB1C3 in DH5alpha competent cells.
Isolated the DNA and put it on a 0,8% agarose gel.
Week 2:
Repeated the transformation of the fusion PCR product with pSB1C3.
Doing the nitrocefin activity test.
Received the first IDT shipment with our GIGA-BLOCKs (gBLOCKs) containing our Zincfinger arrays fused to N-/C-TEV through a GSAT-linker.
Week 3:
Annealing of the gBLOCKS.
Cloning PCR fusion product in pUC35.
Transformed the PCR fusion product with pUC35 into D5alpha competent cells.
Isolated the PCR fusion product with pUC35 and sent it for sequencing.
Ligation and transformation of gBLOCK with pSB1A3.
Ligation and transformation of gBLOCK with pSB1T3.
Week 4:
Sequence results came in. The product was not visible.
Ligation and transformation of gBLOCK with pSB1A3.
Doing the TEV cleaving assay.
Running SDS-PAGE from TEV and BRCA-F6.
Doing the nitrocefin activity test again to see if the nitrocefin is still stable.
September
Week 1:
We came to the conclusion that our DNA-bridges will never arrive and are being held at Customs. We had no money left to order from IDT and ordering again from Twist Bioscience will take too long.
Week 2:
Talks with stakeholders for the future of HRDK and the replacement of nitrocefin with a more friendly substance.
Doing the TEV cleaving assay again.
Week 3:
Finishing up labwork surrounding PCR of the gBLOCKs and transformation of the gBLOCKs into pSB1C3 and pSB1T3.
Week 4:
Resuspending and transforming the purple and pink chromoproteins from iGEM team Upsala 2013.
October
Week 1:
Superbatch was made of the purple and pink chromoproteins.
Experimental tests were performed with chromoproteins in different fluid conditions which vary in pH-conditions to follow absorbance development.
