21/08/18

The Cu-promotor (pSB1K3) and RFP (pSB1K3) were transformed from the kit according to the protocol. The bacteria were plated in 100% and 500%. The custom receptor was ordered and put in the pBSK vector

22/08/18

Bacteria grown and colonies were picked and inoculated in 5 ml LB with

• Cu-promotor (pSB1K3) = Kanamycin
• RFP (pSB1K3) = Kanamycin
• Custom Receptor pBSK = Ampicillin

23/08/18

Miniprep was performed according to the protocol

• Cu-promotor → 250
• Custom receptor → 360
• RFP → 91.7

The “dirty” method of cloning was used to couple the Cu-Promotor and RFP in one vector. The custom receptor was transformed to another vector (pSBK)

For each of the three samples a mastermix was made. For each digestion the DNA concentration was reduced to 25 ng/ul. 4 ul of sample was transferred to a PCR tube with 4 ul of the according restriction mix. The samples were incubated for 1 hour at 37 C. After 1 hour the samples were taken out of the incubator and enzymes were heat inactivated at 80C for 20 min. 1ul of Cu-promotor and 1ul of RFP were added to one PCR tube. Furthermore 1ul DNA T4 ligase buffer and 0.5 ul of T4 ligase were added. The total volume was made up to 10 ul with 3.5 ul dH2O. The samples were incubated for one hour (RT). Enzymes were deactivated with heat kill 80C 20 min.

DNA was transformed in DH5a according to the NEB protocol.

24/08/18

Transformation was successful. Colonies were picked for colony PCR to identify whether the right construct was transformed. 8 colonies of each plate were selected (Cu-promotor + RFP 100%, Cu-promotor + RFP 500%, Custom receptor 100, Custom Receptor 500). Each colony was incubated in PCR tubes with 50 ul LB without antibiotic for 1 hour.

27/08/18

Samples from prior colony PCR were analysed with a 0.8% Agar gel electrophoresis.

28/08/18

The results obtained in the gel electrophoresis DNA analysis (27/8/18) was not satisfactory. Therefore four new colonies were picked for colony PCR and put in 50 µl LB and incubated for 1 hour from each of the following plates: Cu + RFP 100%, Cu + RFP 500%, Custom Receptor 100%, Custom Receptor 500%. 2 µl was of each pick was transferred to new PCR tubes with the 18 µl of following mix:

29/08/18

The DNA of the (28/08/18) samples was purified according to the miniprep protocol.

The prior prepared clonation (28/08/18) was transformed in DH5α according to the iGEM protocol.

30/08/18

No colonies of the transformation(29/8/18) were observed on the petri dishes.

The purified DNA of samples 4,12,14 (29/8/18) were send for sequencing with forward and reverse primer.

Custom receptor in PBSK3

05/09/18

Samples from sequencing were analysed. Custom receptor (12) was placed in the PBSK3 backbone correctly. The sequence of Cu + RFP was difficult to interpret. Therefore Cu + RFP (4) was incubated again in 5 ml LB + Ampicillin from the safe overnight.

06/09/18

The Cu + RFP DNA was purified according to the protocol with minor modifications. The bacteria were split up in fractions of 1.5 ml and pelleted at full speed (21 000 RCF) for one minute. Supernatant was discarded and 150 µl buffer A1 was added. The pellets were resuspended and 250 µl lysis buffer was added. The eppendorfs were tilted 5 times and incubated for 2 min. 450 µl buffer A3 was added and tilted till the blue color disappeared. The fractions were pelleted (21 000 RCF 1 min). The supernatant of all three samples was loaded on one column. The rest of the experiment was performed as described in the protocol. The DNA was eventually solubilised in 20 µl elution buffer at 60 degrees celsius. The final concentration was 463 µg/ml. The sample was sent for sequencing in a 500 ng concentration with VF and VR primer.

10/09/18

The sequence of Cu + RFP did not contain the copper promotor, only RFP.

11/09/18

New method of cloning was tried. We wanted to place the RFP into the Cu promotor PSBK backbone. To do this, digestion was performed according to the NEB digestion protocol. The cu promotor was cut with SpeI and PstI, RFP was cut with Xbal and PstI. After heat inactivation the samples were placed in the -20 freezer

12/09/18

The prior cut samples were ligated using the NEB protocol. 2.4 µl RFP was used as insert with 1 µl of Cu promotor backbone. After heat inactivation the sample was transformed according to the iGEM protocol in DH5α and plated on Ampicillin plates.

13/09/18

18/09/18

16 colonies were picked for colony PCR using one-Taq polymerase and a safe of these colonies was made. The PCR samples were loaded on a 0.8% agarose gel. The gel did not contain a successful cloning sample.

24/09/18

We decided to use another approach,since our previous attempts didn’t yield any results. The copper promoter gBlock was cut with the restriction enzymes EcoRI and SpeI. Simultaneously, the RFP vector was cut with EcoRI and XbaI. Both reactions were done overnight in NEB Buffer 2.1.

25/09/18

In the morning the reaction mix was inactivated by heating it to 80 degrees for 20 minutes. A ligation was set up with the fragments, and 9 µl of the ligation product was transformed to 100 µl of competent Dh5a.

26/09/18

The transformation was successful. 6 colonies could be spotted. These were inoculated overnight in 5 ml LB with chloramphenicol.

27/09/18

The DNA of the inoculated cultures was minipreped according to the miniprep protocol. After the first spin down we noticed that the bacterial pellet of one of the colonie number 6 was slightly red. Because it is known that RFP expressing bacteria turn red even without excitation, we concluded that it was most likely that this colony expressed RFP, and thus that the transformation worked. After the isolation of the DNA, a colony PCR was done with the VF and VR primer. The total size of the band that we expected was 824+156 -6 = 974 bp for the fragment + 271 bp for the part outside the BB_sites generated by the VF and VR primer. The total size of the fragment should thus be 1245 bp. The fragment in lane 6 lies between the 1000 bp band and the 1500 bp band and starts slightly lower than the middle, indicating that it is most likely a band of correct size.

The DNA of this colony was send for sequencing.

29/09/18

The sequencing results showed that the construct was correct. Since both the promoter::RFP fusion, and the Custom receptor were now available to us on plasmid backbones with different resistance genes, both constructs were transformed to E. coli Dh5a.

30/09/18

The transformation was successful. A stock solution of 50 mM CuSO4 was made for experiments with the newly transformed copper sensitive bacteria. 2 colonies were inoculated overnight for further experiments.