08 Jakob, Patrick, Eva After moving into our new lab and settling in, first practical steps like aliquoting our primers or preparing LB-Medium and LB-Agar were taken. 09 Jakob, Patrick, Eva We amplified Cas 3 out of E.Coli gDNA via PCR (Q5 Polymerase) and transformed the biobricks BBa_K608351 (K2), BBa_K091001 (K5), BBa_I13453 (K10), BBa_K584000 (AraC Promotor), BBa_K117008 (LsrR Promotor), BBa_R0073 (Mnt Promotor) in competent E. Coli DH5-alpha (NEB iGEM Kit). 10 Eva, Patrick We amplified Cascade out of E.Coli MG1655 gDNA via PCR (Q5 Polymerase) and checked the Cas 3 and Cascade PCRs via agarose gel electrophoresis. (figure 1) We picked colonies for all but the Ba_K608351 (K2) transformations (no growth) for inoculating overnight cultures and made a new batch of competent E.Coli Dh-5α cells. 11 Jakob, Eva, Patrick By Miniprep (Qiagen) we extracted the DNA of the overnight cultures. Amplification via PCR (Q5 Polymerase) of Cascade was repeated (figure 2). BBa_K608351 (K2) was transformed again and the competent cells tested.

15 Jakob Patrick Eva Luzi The biobricks were transformed again. Cascade and Cas 3 PCR products were sent off for Sanger sequencing. 16 Patrick, Eva Since there repeatedly weren’t any colonies for BBa_K608351 (K2), we transformed it in provided NovaBlue™ competent cells. Overnight cultures of the other biobricks as well as an overnight culture of E.Coli Nissle were inoculated. 17 Eva Patrick Katharina Glycerol stocks of the overnight cultures were prepared. Competent E. Coli Nissle cells were produced and the growth of E. Coli Nissle was analysed preliminarily. After Miniprep (Qiagen) of BBa_K516030 (RFP) a PCR (Pfu Polymerase) was run. After digestion of the PCR products Cas 3 and Cascade as well as the plasmid BBa_I13453 (K10), K10_Cascade and K10_Cas3 were ligated. 18 Eva Patrick The K10_Cascade and K10_Cas3 ligations were digested (single) and analysed by gel electrophoresis, together with the RFP PCR product (figure 3). The gel did not show a result for K10_Cascade and K10_Cas3. RFP (PCR Product) and BBa_R0073 (Mnt Promotor) were digested and ligated. 19 Eva Patrick Digestion and Ligation of Cascade, Cas3 and K10 were repeated with increased amounts of DNA. K10_Cascade, K10_Cas3 and BBa_R0073(Mnt)_RFP were transformed.

23 Eva Jakob Patrick Preparation of LB-Medium, LB-Agar and Chloramphenicol plates. Overnight cultures for K10_Cascade, K10_Cas3 and BBa_R0073(Mnt)_RFP colonies were inoculated. After double digestion of BBa_K584000 (AraC Promotor), BBa_K117008 (LsrR Promotor), the fragments pAraC and pLsrR were extracted from the gel. 24 Eva Patrick After Miniprep of K10_Cascade, K10_Cas3 and BBa_R0073(Mnt)_RFP (Qiagen) the plasmids were single digested. Gelelectrophoresis showed the empty backbone (figure 4). The isolated fragments pAraC and pLsrR were ligated into the backbone psB1K3 after digestion. psBIK3_pAraC and psBIK3_pLsrR were transformed. 25 Eva Overnight cultures for psBIK3_pAraC and psBIK3_pLsrR colonies were inoculated. 26 Eva After Miniprep (Qiagen) of psBIK3_pAraC and psBIK3_pLsrR, the plasmids were digested. Gel electrophoresis showed successful ligations (figure 5).

30 Patrick, Katharina The team moved into a new laboratory. The synthesized sequences (K0, K3, K4, K6, K7, K15) arrived and were aliquoted. Chloramphenicol plates were prepared. Cascade, Cas3, BBa_I13453 (K10), RFP and BBa_R0073 (Mnt Promotor) digestions and ligations were repeated and transformed. 31 Patrick Jakob Overnight cultures for K10_Cas3 colonies were inoculated, there was no growth of the other transformations. A PCR (Pfu polymerase) was run on the synthesized constructs, it wasn’t successful. The Minipreps of psBIK3_pAraC and psBIK3_pLsrR were used for retransformation. 01 Patrick The K10_Cas3 overnight culture was miniprepped. Overnight cultures for psBIK3_pAraC, psBIK3_pLsrR, as well as K10_Cascade and BBa_R0073(Mnt)_RFP (some late colonies grew) were inoculated. 02 Patrick Glycerol stocks for psBIK3_pAraC were prepared, there was no growth in psBIK3_pLsrR overnight cultures. After Miniprep of rpsBIK3_pAraC, K10_Cascade and BBa_R0073(Mnt)_RFP the plasmids (also K10_Cas3) were digested and a gel ectrophoresis was run. It showed the transformation of empty backbone in K10_Cascade and BBa_R0073(Mnt)_RFP, successful ligation for psBIK3_pAraC and XY for K10_Cas3 (figure 6).

05 Patrick Eva Antonia PCR (Q5 polymerase) and PCR (Paq polymerase) was run on the synthesized constructs, they weren’t successful. Cascade and Cas3 were successfully amplified via PCR(Q5). 06 Eva Patrick Antonia A gradient PCR (Q5) was run on the constructs. It was discovered that the currently used agarose was falsely prepared with water instead of TAE-Buffer and was discarded. Cascade, Cas3 and BBa_I13453 (K10) were digested and ligated. 07 Patrick Eva Luzi Antonia A new PCR (Q5) was run on the synthesized constructs, gelectrophoresis suggested positive apmlification (figure 7). By amplification with accordingly designed primer, K0 has been added a TetR promotor and is now K1. Due to a technical malfunction in the building further work was delayed.

12 Patrick Katharina After respective digestion and DNA clean up, psBC13_K1, psBC13_K3, psBC13_K6, K5_K4, K10_Cas3, K10_Cascade and BBa_R0073(Mnt)_RFP were ligated. 13 Patrick Eva Antonia Marie Yesterday’s ligations were transformed. 14 Luzie Marie Eva A colony PCR (Taq polymerase) was run on psBC13_K1, but the gelelectrophoresis was negative (figure 8). Overnight cultures of psBC13_K1, psBC13_K3, psBC13_K6, K5_K4, K10_Cas3 and K10_Cascade colonies were inoculated. 15 Marie Patrick Eva After Miniprep the plasmids were double digested. Gel electrophoresis suggested successful ligations of psBC13_K6, K10_Cas3, K10_Cascade and BBa_R0073(Mnt)_RFP (figure 9). 16 Patrick Miniprep of psBC13_K1 was digested but gelectrophoresis was negative. Cascade and Cas3 were amplified by PCR (Q5 polymerase). The synthesized construct K2 arrived, was diluted and amplified via PCR (Q5 polymerase).

19 Patrick Jakob Eva After respective digestion and DNA clean up, psB1C3_K1, psB1C3_K3, psB1C3_K7 and K5_K4 were ligated. The BBa_R0073(Mnt)_RFP plasmid was digested to isolate Mnt_RFP by gel extraction (figure 10). The backbone pSB1K3 was digested with respective enzymes and pSB1K3_Mnt_RFP was ligated. K1, K2, K3, K4, K7 and K15 were amplified via PCR (Q5 polymerase) with a new programm. 20 Eva Patrick Last weeks psBC13_K6, K10_Cas3 and K10_Cascade were sent off to Sanger sequencing. Yesterday’s ligations were transformed as well as K10_Cascade retransformed. 21 Marie Zoe Patrick A new batch of competent cells was prepared and overnight cultures of psB1C3_K1, psB1C3_K7, K5_K4 and K10_Cascade inoculated. 22 Patrick Eva Sanger Sequencing was positive on K10_Cascade. Chloramphenicol and Kanamycin plates were prepared. After Miniprep of psB1C3_K1, psB1C3_K7, K5_K4 and K10_Cascade the plasmids were digested and run on a gel. Digestions of psB1C3_K1 and K5_K4 were negative. The new competent cells were transformed with psB1C3_K3, psB1K3_Mnt_RFP and iGEM competent cell test plasmids. 23 Marie Patrick The constructs K1, K2, K3, K4, K7 and K15 were amplified by PCR (Q5 polymerase). Double digestion of psB1C3_K7 and K10_Cascade was repeated. The gel electrophoresis suggested XY (figure 11).