Weekly Journal

Week 1 --- 04.08.14 - 08.08.14

In the first week (04.08.14 - 08.08.14) we successfully transformed E.coli (XL-10-Gold) with our all received parts. pUC57- Gal and pUC57-NAGA, containing two of our Enzymes came in a shipping from Genscript. pRSH10.1-SNAP, containing the SNAP-Tag was a donation from a working group at our own institute. We also recieved pEX-A2-Intein, containing our N-split-intein from Eurofins. We minipreped the constructs and controlled them by restriction. We also transformed XL-10-Gold with pSB1C3-SpyCatcher, which we got from the registry, but got a lawn on the plate. It turned out that the used XL-10-Gold did already have a resistance against chloramphenicol, which is selected for in pSB1C3. Besides that, we ran tests with several lab devices, including the gel documentation system where we also tested out our DNA ladder and our DNA-Stain-G, which we were not confident in and decided to switch to Midori Green instead.

Week 2 --- 11.08.14 - 15.08.14

In week two and three our 4th semester bachelor students attended a bachelor module, which we specifically organised with professors at the institute to improve our skills at cloning methods. We thought this was a important step, as our team mostly consists of 4th semester Bachelor students with little experience in longer projects. The transformed constructs pUC57- Gal, pEX-A2-Intein, pRSH10.1-SNAP and pUC57-NAGA from last week were amplified in E.coli and purified, from where we subcloned them into pSB1C3, which we intended as our assembly vector aswell as out vector of choice for registry parts. We also tried to do three-part-assemblies in pSB1C3 with following inserts: SNAP+NAGA, Intein+NAGA, Intein+aGal and SNAP+aGal. Unfortunately we didn’t succeed in the transformation of E.coli BL21 DE3 with our ligated constructs. Possible reasons for that were thought to be improper concentrations of the parts for the ligations. Our last part, EABase-pEX-K4, arrived in the second week and was transformed into E.coli XL 10 Gold.

Week 3 --- 18.08.14 - 22.08.14

In the 3rd week we made a batch of competent BL21DE3. We received the BL21DE3 stock from a working group at our institute. Then we retried the ligation of several parts with pSB1C3, namely our three enzymes (NAGA, Gal and EABase) each with Intein (three-part-assemblies), aswell as our enzymes with of the three enzymes with pSB1C3-Spy(two-part-assembly) and all of our tags and enzymes with pSB1C3(two part assembly) except for Spy-Tag, which already was in pSB1C3. All the ligated constructs were transformed into E.coli BL21DE3. Although this time we used higher concentrations of our parts for the ligation, it didn’t work either. In order for the SNAP-Tag to be used, we had to add the RFC25 prefix and suffix to it by overhang PCR.

Week 4 --- 25.08.14 - 29.08.14

After the transformations into BL21DE3 failed multiple times we decided to swich to a different strain. We received a sample of competent NEB5a from a group at our institute from which we made a batch of our own competent cells. We tried to ligate our parts with pSB1C3 but this time we added extra ATP to the mix and used a decreasing temperature gradient for ligation. With the new competent NEB5a, we finally succeeded in transforming the ligated constructs pSB1C3-Intein, pSB1C3-NAGA, pSB1C3-Intein-NAGA, pSB1C3-aGal and pSB1C3-Intein-EABase. Furthermore we modified our chosen expression vector (pETBlue1), by ligating it with annealed oligonucleotides.

Week 5 --- 01.09.14 - 05.09.14

Minipreparation and subsequent control restriction of the following constructs in pSB1C3 confirmed last week’s transformations: aGal, NAGA and EABase each with Spy and SNAP-NAGA, SNAP-EABAse. We also confirmed the modified pETBlue1, named pETBlue1.1 by control restriction and transformed NEB5a with it. pETBlue1.1 was purified and used for ligation with our one-part and two-part-inserts. However, the transformation of the ligation constructs in NEB5a did not work. Apart from that, we aliquoted our pSB1C3-constructs, that had previously been confirmed by digestion and gel electrophoresis, to send them to GATC-biotech for sequencing. Results were in the next day, with confirmation of correct inserts. Although sequencing did not cover the whole insert, one could see that the parts were ligated correctly.