Team:Groningen/Template/MODULE/Notebook/toolbox

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 at THIS PAGE LINKLINKLINK. 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 THIS PAGE LINKLINK. 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 BBa_K1033219 (CP1), BBa_K1033220 (CP8), BBa_K1033221 (CP11), BBa_K1033222 (CP29), BBa_K1033223 (CP30), BBa_K1033224 (CP41) and BBa_K1033225 (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 THIS PAGE LINKLINK. The primers are denoted with the position of the illegal restriction site that they should remove. Illegal EcoRI sites are abbreviated with an E, XbaI sites with an X, SpeI site with an S and PstI sites with a P.

The CP promoters in pSB1C3, that were isolated in the week of 14 - 20 Juli, were tested on their insert size. For this, a PCR was done on the plasmid using the primers VF2 (BBa_G00100) and VR (BBa_G00101) were used. 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 Juli - 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. The white colonies were tested on insertsize with a colony PCR with the VF2 (BBa_G00100) and VR (BBa_G00101) 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 (BBa_G00100) and VR (BBa_G001001). The sequence of each BioBrick was confirmed.

Colony PCR on more transformants containing pSB1C3 with NisB and NisRK was repeated, using VF2 (BBa_G00100) and VR (BBa_G00101). 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. No positive transformants were found.

A PCR was done on all the genes that contained illegal restriction sites: NisI, NisT, NisP and NisFEG. This should generate multiple PCR fragments for each gene, that will be assembled again using Gibson assembly. The PCR was split into two seperate reactions because of the large differences between the size of the different fragments. Fragments were sorted on < 750 kb and > 750 kb. Experiment not continued because of time limits.

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 with VF2 (BBa_G00100) and VR (BBa_G00101) primers and the a restriction analysis was done using EcoRI and PstI. Cultures that showed a correct insert size were sent for sequencing.

SEQUENCING RESULTS

A PCR was done to generate fragments for assembling genes that had illegal restriction sites. Every fragment was succesfully amplified. The fragments were purified using the GeneJET PCR clean up kit. were not fully clean, so only the fragments for the combined BioBrick of NisF, NisE and NisG was made using Gibson assembly. The transformants that contained pSB1C3 with NisFEG were tested using colony PCR. No positive transformants were found.

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

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.

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.

A PCR was done on all the genes that contained illegal restriction sites: NisI, NisT, NisP and NisFEG. This should generate multiple PCR fragments for each gene, that will be assembled again using Gibson assembly. The PCR was split into two seperate reactions because of the large differences between the size of the different fragments. Fragments were sorted on <750 kb and >750 kb. The experiment was not continued because of time limits.

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.

The sequences of NisA, NisC, NisRK and sfGFP(Bs) were analyzed. The sequences of NisA and sfGFP(Bs) were completely correct. The sequence of NisC showed that a mutation occured at residue 185, where an AAT was changed to an AAA, changing an asparagine to a lysine. The changed amino acid was on the outside of the protein, so may not have an effect on the function of NisC. The sequence of NisRK showed two mutations. At residue 199 of NisR a GAA was changed to a GAG, not changing the amino acid at this position. At residue 300 of NisK an ACA was changed to an ATA, changing a threonine to an isoleucine. The PNisI with RBS showed a large mutation. At position 508, 18 basepairs were missing. Therefore it was decided to not send PNisI to iGEM HQ anymore.

A PCR was done to generate fragments for assembling genes that had illegal restriction sites. Every fragment was succesfully amplified. The fragments were purified using the GeneJET PCR clean up kit. Not all fragments have a sufficient concentration or purity, so only the fragments for the combined BioBrick of NisF, NisE and NisG was made using Gibson assembly. The transformants that contained pSB1C3 with NisFEG were tested using colony PCR. No positive transformants were found.

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