Team:Groningen:Notebook:Protocols

Primers were designed for making BioBricks out of the separate genes from the nisin operon. For this, the sequence of the transposon Tn5307 was used, a transposon that contains the nisin operon, and of which the sequence is known.¹ This sequence is close to the sequence of the transposon Tn5276, the transposon which will form the template for making the new BioBricks. The primers for making a BioBrick out of PNisI with a RBS were based on a sequence found in a research paper that documented the identification of this promoter.² The list of primersequences can be found in the first table at the bottom of this page. It was decided to combine the genes NisR and NisK as one BioBrick and NisF, NisE and NisG as one BioBrick, because these genes are related in function or work together.

There were also primers designed for making a BioBrick out of the sfGFP(Bs), the primersequences can be found at second table at the bottom of this page. This sfGFP was originally optimized for Bacillus subtilis. When it was tested in Lactococcus lactis, the sfGFP(Bs) was shown to perform really well in L. lactis as well.³

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.

The CP promoters in pSB1C3, that were isolated in the week of 14 - 20 July, were tested on their insert size. For this, a PCR was done on the plasmid using the primers VF2 and VR. The insert size corresponded to the expected size.

A touchdown PCR was done on the genes of the nisin operon and the sfGFP(Bs) gene. In this PCR the annealing temperature dropped from 55 °C to 45 °C, lowering the temperature with 1 °C each cycle. This unfortunately resulted in a PCR program of just 10 cycles, instead of the intended 30. The remaining steps of the program were finished the next morning. Only the genes NisA, PNisI and sfGFP(Bs) were amplified this way, see figure 1. Therefore, the PCR was repeated, this time with a complete cycle. No additional genes were amplified this way.

Another attempt was made for amplification of the remaining genes. This time, the most ideal annealing temperature for each gene was used by using a gradient PCR and placing the tubes at the optimal temperature. This still did not amplify the remaining genes. Also, a PCR with a general annealing temperature of 50 °C was done. This also did not amplify the remaining genes.

The genes that were amplified in the first PCR (NisA, PNisI and sfGFP(Bs)) were purified using the GeneJET PCR Purification Kit from Thermo Scientific. The purified products were digested with the enzymes EcoRI and PstI, using 2 μl of the product. These purified and restricted products were loaded on gel, see figure 2. It was then discovered that the genes were barely visible on gel. The restricted genes were purified with the GeneJET PCR Purification Kit and the concentration was measured with the NanoDrop 1000. The clean, restricted products were too low in concentration to be suitable for ligation.

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.