Team:Groningen/Template/MODULE/Notebook/toolbox

A collection of constitutive promoters was also desirable for the toolbox. We decided to use the CP promoter collection and test it in Lactococcus lactis, that was BioBricked by the Uppsala iGEM team of 2013. The CP promoters are registered as CP1, CP8, CP11, CP29, CP30, CP41 and CP44. These parts were ordered from iGEM HQ, except for CP8 as for this promoter the sequencing was inconsistent. The promoters were sent by iGEM HQ using Escherichia coli containing pSB1C3 with the promoter as insert. E. coli was grown and the plasmids with the promoter were isolated.

BioBricking the genes NisT, NisI, NisP and the combined genes NisF, NisE and NisG gave problems with illegal restriction sites.
The illegal restrictionsite in NisI could be removed using the reverse primer as listed above. This primer contains a mutation needed to remove the illegal restriction site.
The other parts gave more problems, as these all contained multiple restriction sites. Therefore, it was decided to use Gibson assembly with primers containing mutations to remove the restriction site. By using Gibson assembly, the genes could also be constructed in pSB1C3 in one go. The primersequences can be found at the third table at the bottom of this page.

Because the PCR products of NisA, PNisI and sfGFP(Bs) were lost during the purification and restriction in the week of 28 July - 3 August, the PCR was repeated for this genes, together with the genes that could not be amplified yet. This time a PCR was used that did not lower in temperature, like the touchdown PCR, but that increased in temperature with each step. This way it was hoped to get over the huge gap between the annealing temperature of the primer in the first cycle and the annealing temperature of the primer when the flap of the primer can also anneal to the first PCR products. The temperature was set to increase from 40 °C to 60 °C in 20 cycles. Then, an additional 20 cycles were done at 65 °C. The PCR was performed under standard conditions as was done before, together with a series of PCR that contained GC buffer (supplied with the Phusion DNA polymerase by Thermo Scientific), and a series of PCR with GC buffer and 1.5% DMSO. This time, with the help of Lisa, the PCR was finally successful for all genes, see figure 3.

The PCR on the BioBrick of the combined genes NisR and NisK was repeated because the yield of this product was very low after the PCR in the week of 4 - 10 August. The mixture of the previous PCR reaction was used as the template. The PCR was performed under standard conditions, with an annealing temperature of 64 °C and 1 μl, 2μl and 3 μl template. No product was obtained with this PCR. So the PCR was once again repeated, this time using diluted template. The mixture was diluted 50x, 300x, 1500x and 15000x. The PCR was repeated using 1 μl and 2 μl of each of the dilutions. This way, product was obtained for all reactions.

The PCR products of the genes NisA, NisB, NisC, NisRK, PNisI and sfGFP(Bs) were purified using the GeneJET purification kit. Then 1 μg of each purified product was restricted with EcoRI and PstI, together with 1 μg of the pSB1C3 plasmid. The restriction enzymes were then inactivated by heating the samples at 80 °C for 20 minutes. Ligated 6 μl of the restricted PCR products to 2 μl restricted pSB1C3. Inactivated the ligase by incubating at 65 °C for 10 minutes. Mixed 5 μl of the ligation mixture with 25 μl electrocompetent Escherichia coli DH5α. The transformants were grown on LB with 10 μg/ml chloramphenicol, see figure 4. The white colonies were tested on insertsize with a colony PCR with the VF2 and VR primers. For every gene transformants were found that contained pSB1C3 with a correctly sized insert, except for NisB and NisRK.

The E. coli that contained the pSB1C3 plasmid with a correctly sized insert were grown as a liquid culture and the plasmid was isolated using the GeneJET Plasmid Miniprep Kit.

The plasmids containing the new BioBricks NisA, NisC, PNisI and sfGFP(Bs) that were isolated in the week of 11 - 17 August were concentrated with a SpeedVac and then sent for sequencing with the primers VF2 and VR. The sequence of each BioBrick was confirmed.

Colony PCR on more transformants containing pSB1C3 with NisB and NisRK was repeated, using VF2 and VR. This time, a correct insert size wasbfound for a NisRK transformant.
Because there was still no correct insert size found for a NisB transformant, the remaining NisB colonies were tested for a correct insert size. There were no positive transformants found.

Because of the constant low concentrations of the plasmid pSB1C3 with the new BioBricks, it was decided to transform the isolated pSB1C3 with the new BioBricks (NisA, NisC, NisRK, PNisI and sfGFP(Bs)) again in Escherichia coli DH5α. The pSB1C3 plasmids containing the new BioBricks were again isolated. For each transformation, two seperate cultures were grown in LB with 10 μg/ml chloramphenicol. A miniprep was done on the cultures and the insert size was checked with colony PCR, using VF2 and VR as primers. In addition, a restriction analysis was done using EcoRI and PstI. Cultures that showed a correct insert size were sent for sequencing.

Completed BioBricks were sent to iGEM HQ in a pSB1C3 backbone: NisA, NisC, NisRK and sfGFP(Bs).