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Team:Groningen/Notebook/Biobricks
From 2014.igem.org
7 – 13 Juli
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
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 primer designed for making a BioBrick out of the sfGFP(Bs). 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
14 - 20 Juli
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.
21 - 27 Juli
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 primers in the table below 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.
28 Juli - 3 August
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 &mul 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.
4 - 10 August
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.
11 - 17 August
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 &mul, 2&mul and 3 &mul 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 &mul and 2 &mul 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 &mug of each purified product was restricted with EcoRI and PstI, together with 1 &mug of the pSB1C3 plasmid. The restriction enzymes were then inactivated by heating the samples at 80 °C for 20 minutes. Ligated 6 &mul of the restricted PCR products to 2 &mul restricted pSB1C3. Inactivated the ligase by incubating at 65 °C for 10 minutes. Mixed 5 &mul of the ligation mixture with 25 &mul electrocompetent Escherichia coli DH5&alfa.
18 - 31 August
References
1. Trmčić, A. et al. (2011) Complete nisin A gene cluster from Lactococcus lactis M78 (HM219853) – obtaining the nucleic acid sequence and comparing it to other published nisin sequences. Genes Genom. 33: 217-221
2. Li, H. and O´Sullivan, D.J. (2006) Identification of a nisI Promoter within the nisABCTIP Operon That May Enable Establishment of Nisin Immunity Prior to Induction of the Operon via Signal Transduction. J. Bacteriol. 188: 8496-8503
3. Overkamp, W. et al. (2013) Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging. Appl. Environ. Microbiol. 79: 6481-6490
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
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.
| Primer | Sequence |
|---|---|
| NisA forward | GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAGTACAAAAGATTTTAA |
| NisA reverse | GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTTGCTTACGTGAA |
| NisB forward | GTTTCTTCGAATTCGCGGCCGCTTCTAGATGATAAAAAGTTCATTTAA |
| NisB reverse | GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATCATTTCATGTATTCTT |
| NisT forward | GTTTCTTCGAATTCGCGGCCGCTTCTAGATGGATGAAGTGAAAGAATTTACATCA |
| NisT reverse | GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTCATCATTATCCT |
| NisC forward | GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAATAAAAAAAATATAAA |
| NisC reverse | GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATCATTTCCTCTTCCCTC |
| NisI forward | GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAGAAGATATTTAATACT |
| NisI reverse | GTTTCTTCCTGCAGCGGCCGCTACTAGTATTACTAATTTCCTACCTTCG |
| NisP forward | GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAAAAAAATACTAGGTTT |
| NisP reverse | GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATCAATTTTTAGTCTTTC |
| NisRK forward | GTTTCTTCGAATTCGCGGCCGCTTCTAGGTGTATAAAATTTTAATAGT |
| NisRK reverse | GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTACTTTTTTATTTTTA |
| NisFEG forward | GTTTCTTCGAATTCGCGGCCGCTTCTAGATGCAGGTAAAAATTCAAAA |
| NisFEG reverse | GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATCTAATCTTTTTTT |
| PNisI+RBS forward | GTTTCTTCGAATTCGCGGCCGCTTCTAGAGAGCACTGGATAATGACTATT |
| PNisI+RBS reverse | TACTAGTAGCGGCCGCTGCAGGAAGAAACTTCCTCTTCCCTCCTTTCAA |
There were also primer designed for making a BioBrick out of the sfGFP(Bs). 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
| Primer | Sequence |
|---|---|
| sfGFP(Bs) forward | GTTTCTTCGAATTCGCGGCCGCTTCTAGATGTCAAAAGGAGAAGAGCT |
| sfGFP(Bs) reverse | GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTACTTATAAAGCTCAT |
14 - 20 Juli
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.
21 - 27 Juli
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 primers in the table below 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.
| Gene | Primer | Sequence |
|---|---|---|
| NisT | Prefix forward | GTTTCTTCGAATTCGCGGCCGCTTCTAGATGGATGAAGTGAAAGAATTTACATCA |
| S980 reverse | TTGTTCCATAAACAAACTTGTATTATAAATGATGTAAATA | |
| S980 forward | TATTTACATCATTTATAATACAAGTTTGTTTATGGAACAA | |
| S1210 reverse | CCATAGTTGGTTGATATAATCCTGAAATTATCTTTACTTGTGTAC | |
| S1210 forward | GTACACAAGTAAAGATAATTTCAGGATTATATCAACCAACTATGG | |
| Suffix reverse | GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATTCATCATTATCCT | |
| Suffix forward | ATGAATAATAATACTAGTAGCGGCCGCTGCAGGAAGAAACAAAAGGGCAA | |
| Prefix reverse | CACTTCATCCATCTAGAAGCGGCCGCGAATTCGAAGAAACATCCTTAGCG | |
| NisP | Prefix forward | GTTTCTTCGAATTCGCGGCCGCTTCTAGGTGAAAAAAATACTAGGTTT |
| P225 reverse | AGTCCTTGTTGTCGATTTTACTGCTGGTGACTGCGCCCCT | |
| P225 forward | AGGGGCGCAGTCACCAGCAGTAAAATCGACAACAAGGACT | |
| E347 reverse | AACGCTATCTCTCTTACTAAATTCAGAACTAACTTGAGTT | |
| E347 forward | AACTCAAGTTAGTTCTGAATTTAGTAAGAGAGATAGCGTT | |
| E1657 reverse | CAATACTCTATTCCCATTAACTTCTGGACTATTCATTAGA | |
| E1657 forward | TCTAATGAATAGTCCAGAAGTTAATGGGAATAGAGTATTG | |
| Suffix reverse | GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATCAATTTTTAGTCTTTC | |
| Suffix forward | AAATTGATAATACTAGTAGCGGCCGCTGCAGGAAGAAACAAAAGGGCAA | |
| Prefix reverse | CCTAGTATTTTTTTCATCTAGAAGCGGCCGCGAATTCGAAGAAACATCCTTAGCG | |
| NisFEG | Prefix forward | GTTTCTTCGAATTCGCGGCCGCTTCTAGATGCAGGTAAAAATTCAAAA |
| P186 reverse | TCCAGTATCAGCAGAAATTAAACCAAACAAAATTTTCATC | |
| P186 forward | GATGAAAATTTTGTTTGGTTTAATTTCTGCTGATACTGGA | |
| E336/X349 reverse | CCTGTTTCTGCCAAACCAATCACTTCTAGTGTTTCATGTATTCTCTTA | |
| E336/X349 forward | TAAGAGAATACATGAAACACTAGAAGTGATTGGTTTGGCAGAAACAGG | |
| Suffix reverse | GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATCTAATCTTTTTTT | |
| Suffix forward | TAGATAATAATACTAGTAGCGGCCGCTGCAGGAAGAAACAAAAGGGCAA | |
| Prefix reverse | TTTTACCTGCATCTAGAAGCGGCCGCGAATTCGAAGAAACATCCTTAGCG |
28 Juli - 3 August
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 &mul 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.
4 - 10 August
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.
11 - 17 August
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 &mul, 2&mul and 3 &mul 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 &mul and 2 &mul 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 &mug of each purified product was restricted with EcoRI and PstI, together with 1 &mug of the pSB1C3 plasmid. The restriction enzymes were then inactivated by heating the samples at 80 °C for 20 minutes. Ligated 6 &mul of the restricted PCR products to 2 &mul restricted pSB1C3. Inactivated the ligase by incubating at 65 °C for 10 minutes. Mixed 5 &mul of the ligation mixture with 25 &mul electrocompetent Escherichia coli DH5&alfa.
18 - 31 August
References
1. Trmčić, A. et al. (2011) Complete nisin A gene cluster from Lactococcus lactis M78 (HM219853) – obtaining the nucleic acid sequence and comparing it to other published nisin sequences. Genes Genom. 33: 217-221
2. Li, H. and O´Sullivan, D.J. (2006) Identification of a nisI Promoter within the nisABCTIP Operon That May Enable Establishment of Nisin Immunity Prior to Induction of the Operon via Signal Transduction. J. Bacteriol. 188: 8496-8503
3. Overkamp, W. et al. (2013) Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging. Appl. Environ. Microbiol. 79: 6481-6490
Notebook / Biobricks
