Difference between revisions of "Team:SKLMT-China/Notebook Overview"
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<p>amplify the chloramphnicol and promoter segment and add vector homology arms via series PCR. Purify the PCR products using the TianGen PCR Purification Kit and elute into 30ul of autoclaved ddH2O. Quantify the elution using a NanoDrop UV spectrophotometer, store them at 4℃。</p> | <p>amplify the chloramphnicol and promoter segment and add vector homology arms via series PCR. Purify the PCR products using the TianGen PCR Purification Kit and elute into 30ul of autoclaved ddH2O. Quantify the elution using a NanoDrop UV spectrophotometer, store them at 4℃。</p> | ||
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<p>transform the plasmid pBBR1-kan-amp-ccdb-BAD-GFP-firefly into p.fluorescence pf-5 via electroporation. </p> | <p>transform the plasmid pBBR1-kan-amp-ccdb-BAD-GFP-firefly into p.fluorescence pf-5 via electroporation. </p> | ||
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<p>Chracterize the strength of different promoters by luciferase firefly assay. </p> | <p>Chracterize the strength of different promoters by luciferase firefly assay. </p> | ||
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<p>modeling </p> | <p>modeling </p> | ||
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<p>constract our software tool </p> | <p>constract our software tool </p> | ||
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Revision as of 14:35, 15 October 2018
Team registration and brainstorm
During this time, we registrated the team SKLMT-China in iGEM. In order to verify the feasibility of our project, we discussed with professors and experts and did some human practice work.
Find promoters with various strength in the light of transcriptom references. Take the sequence from the upstream of a specific gene.
| Number | Promoter | Sequence 5'-3' | Length |
|---|---|---|---|
| 01 | PampC | ACGCCGGGGCCGCAAAGCCGCTGGGACAAACGCGGCGTACATAACCAGAACCCAGGGAAACCAACCAATC | 70 |
| 02 | ParaA | CTACGACGGCTTCCTGACCCTATAGCTCTAGCAACACCCCCGATTGTTTTGTCTCTGGAGGATCACAGTA | 70 |
| 03 | PfabD | TGTCAGTTTCTTGCGTCCACCACGGTGTGACGTCAATTCTCCGACGACAAGATCATTAGGGGCTTGTTAC | 70 |
| 04 | PccmD | AGGTGCTCAAGCGTGAAGCCCGGACCAGTTGGGTCAAGGCCGAAGTCCAACGCAGCCTGGAGGCCGCCCG | 70 |
| 05 | PfliA | CGGCGAACCCACGCGGGCATCTGGAGTTTTTTGTCGAGCGGCTAGTGCATCAAACAGCGGGACCGGTGCT | 70 |
| 06 | PltR | GGCTGTCATGATCTATTTGTAATTTGCCTTTACAAAAATAAGACACTAAAAATTCTAAAAGGATTTAGGA | 70 |
| 07 | PproC | GCGCTGTTTGGTGCCCGCGACTACAGTCAGTCCTGATGCGACTGACCCCGACCTCTTGTAAGGACCTGTC | 70 |
| 08 | PrecA | GCTGTGGAATAATACTGGCTACTTATACAGGTGTTGGCCGTCAGGCCTTATTGATTACGTGAGGACTTTA | 70 |
| 09 | PrpoD | TGAGGTCATAGCTCGGGTATAATCCTCGGCTTGTTTTTTGCCCGCCAAGACCTTCAGTGGATAGGGTGTT | 70 |
| 10 | PrpoS | AGGCTCCAGCGTTGCCAGGGATAAAGGCGCCGCTTGAGCCTGAGGTCGAACTCACCAAAGGACTATAACA | 70 |
| 11 | PdnaA | TAAGTGGGCGGCTGATCGCTACAATGGCCGCTTGTTTTTGCCTCACCGGCTTTCAACTTAGGGGATATCC | 70 |
| 12 | Ppol | ACCTTGTCCCTTTGTCGTCAACGGTCCCGCTATTCTAGCGCCGGACCCGCCGAAAAGGTTGATCCCGCTC | 70 |
| 13 | P16s | CGAGAAATCAAAGATGTAACCAACGATTGCTGAGCCAAGTTTAGGGTTTTCTCAAAACCCAAAGATGTTT | 70 |
| 14 | Psig | TGCCAAATCGATAAAGCCTCAATGACAATGCGACTAATTATCATTAGTGACACTCAGGCAGTCCGCGTCC | 70 |
| 15 | Patf | GTCGCCGGGCGTAAAGTGATCGCCGTTTGCGCGGCCCGATTGTCCGGCCGCCCGAGTAAGGAGTCTTATC | 70 |
| 16 | Pfme | ATGACCGTCTTGCAAAGGCTTGCTACGGCAAGCCTTTTTCTTTTCCGCATGTTTAACATCGAGAATTCCC | 70 |
| 17 | Pmem | GGTTAATCCGGGGTTTGTCGTTTCTGCCCGCCCCGCTATCATTCGCCGCATCTTTCGAGGGGTATGACCG | 70 |
| 18 | Pfat | CGTTTACGGCTAGCGCCCGGAGCCGGGGCTCGGGGCAGGATGCACACATCACTGACCAGAGGATGGAATC | 70 |
| 19 | Pami | GGTTTGGGGTTGCGCACTAACTTGCGCAGTGGCGATATAAGCGCCCTATAACAATTCCAGGGGTCGTTGT | 70 |
| 20 | Prib | TTCGCGCCCTAATTCGTGCGGTATTCAACAATAGTGTTGGGTGGCGGCACGCTAGCCTGAGGAATACACC | 70 |
| 21 | Pabc | CCCGCAAATAAAATTGCCGGCCCAGGCCGGCAGGCGCCCGGGGTCGGGCGACTTACAAGGAGCTTGTTGG | 70 |
| 22 | Pedo | TGAAGGCGCGCTGAGCGTGGCTGACCAGTTGATCGCCAAAGAGCGCGCAGCCAAGTAAGCGCCCGCCGCC | 70 |
| 23 | Ptox | AAAGACGCGGGCCTTGAGTGCTCTAACACCCAAGACCCGCTAACCACAAGCAGCTAAACAGGAGCTGAAT | 70 |
| 24 | Pdiv | GAGCTGACCCGGCGCACCCTCAAGCAAGGTCTGGAACTGCTGGGCCTCAAAGTCCTGGAGCAAATGTAAG | 70 |
| 25 | Plip | AGGTTCTGATGTTTTCCGGGCAAGGCCTGAAGTAAAAAAACCGGGGCTTCGGGCCCACGGGAGAAAAATA | 70 |
design the PAGE-purified oligonucleotides used to amplify the chloramphenicol
amplify the chloramphnicol and promoter segment and add vector homology arms via series PCR. Purify the PCR products using the TianGen PCR Purification Kit and elute into 30ul of autoclaved ddH2O. Quantify the elution using a NanoDrop UV spectrophotometer, store them at 4℃。
In order to obtain an promoter library suitable for cloning, promoter sequence flanked by homology arms was added a chloramphenicol selection marker. This sequence was synthesized by sangon ®.
TABLE 2 Oligonucleotides used for PCR of the DNA segment containing the promoter and chloramphenicol selective marker
| Primer | Sequence 5'-3' | length |
|---|---|---|
| Syn-primer 1 | AATGAATTACAACAGTTTTTATGCAGATATCAATTAATTTGGTTATGTGTGGGAGGGCTA | 60 |
| ampC-2 | TGGGTTCTGGTTATGTACGCCGCGTTTGTCCCAGCGGCTTTGCGGCCCCGGCGTATTAATGACGTTGATCGGCACGTAAG | 80 |
| araA-2 | AGACAAAACAATCGGGGGTGTTGCTAGAGCTATAGGGTCAGGAAGCCGTCGTAGATTAATGACGTTGATCGGCACGTAAG | 80 |
| fabD-2 | GATCTTGTCGTCGGAGAATTGACGTCACACCGTGGTGGACGCAAGAAACTGACAATTAATGACGTTGATCGGCACGTAAG | 80 |
| ccmD-2 | TGCGTTGGACTTCGGCCTTGACCCAACTGGTCCGGGCTTCACGCTTGAGCACCTATTAATGACGTTGATCGGCACGTAAG | 80 |
| fliA-2 | TTTGATGCACTAGCCGCTCGACAAAAAACTCCAGATGCCCGCGTGGGTTCGCCGATTAATGACGTTGATCGGCACGTAAG | 80 |
| pltR-2 | AATTTTTAGTGTCTTATTTTTGTAAAGGCAAATTACAAATAGATCATGACAGCCATTAATGACGTTGATCGGCACGTAAG | 80 |
| proC-2 | AGGTCGGGGTCAGTCGCATCAGGACTGACTGTAGTCGCGGGCACCAAACAGCGCATTAATGACGTTGATCGGCACGTAAG | 80 |
| recA-2 | TCAATAAGGCCTGACGGCCAACACCTGTATAAGTAGCCAGTATTATTCCACAGCATTAATGACGTTGATCGGCACGTAAG | 80 |
| rpoD-2 | AAGGTCTTGGCGGGCAAAAAACAAGCCGAGGATTATACCCGAGCTATGACCTCAATTAATGACGTTGATCGGCACGTAAG | 80 |
| rpoS-2 | TGAGTTCGACCTCAGGCTCAAGCGGCGCCTTTATCCCTGGCAACGCTGGAGCCTATTAATGACGTTGATCGGCACGTAAG | 8f0 |
| dnaA-2 | GAAAGCCGGTGAGGCAAAAACAAGCGGCCATTGTAGCGATCAGCCGCCCACTTAATTAATGACGTTGATCGGCACGTAAG | 80 |
| pol-2 | TTCGGCGGGTCCGGCGCTAGAATAGCGGGACCGTTGACGACAAAGGGACAAGGtATTAATGACGTTGATCGGCACGTAAG | 80 |
| 16s-2 | TGAGAAAACCCTAAACTTGGCTCAGCAATCGTTGGTTACATCTTTGATTTCTCGATTAATGACGTTGATCGGCACGTAAG | 80 |
| sig-2 | AGTGTCACTAATGATAATTAGTCGCATTGTCATTGAGGCTTTATCGATTTGGCAATTAATGACGTTGATCGGCACGTAAG | 80 |
| atf-2 | CGGGCGGCCGGACAATCGGGCCGCGCAAACGGCGATCACTTTACGCCCGGCGACATTAATGACGTTGATCGGCACGTAAG | 80 |
| fme-2 | AAACATGCGGAAAAGAAAAAGGCTTGCCGTAGCAAGCCTTTGCAAGACGGTCATATTAATGACGTTGATCGGCACGTAAG | 80 |
| mem-2 | AAGATGCGGCGAATGATAGCGGGGCGGGCAGAAACGACAAACCCCGGATTAACCATTAATGACGTTGATCGGCACGTAAG | 80 |
| fat-2 | CAGTGATGTGTGCATCCTGCCCCGAGCCCCGGCTCCGGGCGCTAGCCGTAAACGATTAATGACGTTGATCGGCACGTAAG | 80 |
| ami-2 | TTGTTATAGGGCGCTTATATCGCCACTGCGCAAGTTAATGCGCAACCCCAAACCATTAATGACGTTGATCGGCACGTAAG | 80 |
| rib-2 | TAGCGTGCCGCCACCCAACACTATTGTTGAATACCGCACGAATTAGGGCGCGAAATTAATGACGTTGATCGGCACGTAAG | 80 |
| abc-2 | AAGTCGCCCGACCCCGGGCGCCTGCCGGCCTGGGCCGGCAATTTTATTTGCGGGATTAATGACGTTGATCGGCACGTAAG | 80 |
| edo-2 | TTGGCTGCGCGCTCTTTGGCGATCAACTGGTCAGCCACGCTCAGCGCGCCTTCAATTAATGACGTTGATCGGCACGTAAG | 80 |
| tox-2 | GCTGCTTGTGGTTAGCGGGTCTTGGGTGTTAGAGCACTCAAGGCCCGCGTCTTTATTAATGACGTTGATCGGCACGTAAG | 80 |
| div-2 | GACTTTGAGGCCCAGCAGTTCCAGACCTTGCTTGAGGGTGCGCCGGGTCAGCTCATTAATGACGTTGATCGGCACGTAAG | 80 |
| lip-2 | GCCCGAAGCCCCGGTTTTTTTACTTCAGGCCTTGCCCGGAAAACATCAGAACCTATTAATGACGTTGATCGGCACGTAAG | 80 |
Set up the PCR reaction using pR6K-cm-ccdB as a template. Typically, set up the PCR reaction (50ul in total) to obtain sufficient amounts of DNA.
| Component | Amount(ul) | Final |
|---|---|---|
| Autoclaved ddH2O | 22 | |
| 2×primeSTAR max premix | 25 | 1× |
| Template, 50ng ul-1 | 1 | 50 ng |
| Syn-promoter-1 Oligo(10uM) | 1 | 30 pmol |
| promoter-2 Oligo(10uM) | 1 | 30 pmol |
| Total | 50 |
Carry out the PCR in a thermal cycle following the instructions below:
| Cycle number | Denaturation | Annealing | Termination | |
|---|---|---|---|---|
| 1 | 94℃,4min | |||
| 2~31 | 98℃, 15s | 59℃, 15s | 72℃, 15s | |
| 32 | 72℃, 4min | |||
| 33 | 10℃, hold |
Add vector homology arms to the PCR products purified
Using the purified PCR products from last step as a template. Use the PAGE-purified oligonucleotides ,Syn-promoter-3 and promoter-4 (Table3), to amplify the cm-promoter.
TABLE 3 Oligonucleotides used for PCR of the DNA segment containing the promoter and chloramphenicol selective marker
| Primer | Sequence 5'-3' | length |
|---|---|---|
| Syn-primer 3 | AATGAATTACAACAGTTTTTAT | 22 |
| ampC-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATGATTGGTTGGTTTCCCTGGGTTCTGGTTATGTACGC | 76 |
| araA-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATTACTGTGATCCTCCAGAGACAAAACAATCGGGGGTG | 76 |
| fabD-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATGTAACAAGCCCCTAATGATCTTGTCGTCGGAGAATT | 76 |
| ccmD-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATCGGGCGGCCTCCAGGCTGCGTTGGACTTCGGCCTTGA | 76 |
| fliA-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATAGCACCGGTCCCGCTGTTTGATGCACTAGCCGCTCG | 76 |
| pltR-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATTCCTAAATCCTTTTAGAATTTTTAGTGTCTTATTTT | 76 |
| proC-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATGACAGGTCCTTACAAGAGGTCGGGGTCAGTCGCATC | 76 |
| recA-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATTAAAGTCCTCACGTAATCAATAAGGCCTGACGGCCA | 76 |
| rpoD-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATAACACCCTATCCACTGAAGGTCTTGGCGGGCAAAAA | 76 |
| rpoS-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATTGTTATAGTCCTTTGGTGAGTTCGACCTCAGGCTCA | 76 |
| dnaA-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATggatatcccctaagttgaaagccggtgaggcaaaaa | 76 |
| pol-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATgagcgggatcaaccttttcggcgggtccggcgctag | 76 |
| 16s-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATaaacatctttgggttttgagaaaaccctaaacttgg | 76 |
| sig-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATggacgcggactgcctgagtgtcactaatgataatta | 76 |
| atf-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATgataagactccttactcgggcggccggacaatcggg | 80 |
| fme-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATgggaattctcgatgttaaacatgcggaaaagaaaaa | 76 |
| mem-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATcggtcatacccctcgaaagatgcggcgaatgatagc | 76 |
| fat-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATgattccatcctctggtcagtgatgtgtgcatcctgc | 76 |
| ami-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATacaacgacccctggaattgttatagggcgcttatat | 76 |
| rib-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATggtgtattcctcaggctagcgtgccgccacccaaca | 76 |
| abc-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATccaacaagctccttgtaagtcgcccgaccccgggcg | 76 |
| edo-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATggcggcgggcgcttacttggctgcgcgctctttggc | 76 |
| tox-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATattcagctcctgtttagctgcttgtggttagcgggt | 76 |
| div-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATcttacatttgctccaggactttgaggcccagcagtt | 76 |
| lip-4 | ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATtatttttctcccgtgggcccgaagccccggtttttt | 76 |
Using the purified PCR products from step2 as a template. set up the PCR reaction (50ul in total) to obtain sufficient amounts of DNA
| Component | Amount(ul) | Final |
|---|---|---|
| Autoclaved ddH2O | 22 | |
| 2×primeSTAR max premix | 25 | 1× |
| Template, 50ng ul-1 | 1 | 50 ng |
| Syn-promoter-3 Oligo(10uM) | 1 | 30 pmol |
| promoter-4 Oligo(10uM) | 1 | 30 pmol |
| Total | 50 |
Carry out the PCR in a thermal cycle following the instructions below:
| Cycle number | Denaturation | Annealing | Termination | |
|---|---|---|---|---|
| 1 | 94℃,4min | |||
| 2~31 | 98℃, 15s | 52~59℃, 15s, 15s | 72℃, 15s | |
| 32 | 72℃, 4min | |||
| 33 | 10℃, hold |
Purify the PCR products using the TianGen PCR Purification Kit and elute into 30ul of autoclaved ddH2O. Quantify the elution using a NanoDrop UV spectrophotometer.
transform the plasmid pBBR1-kan-amp-ccdb-BAD-GFP-firefly into GB05 red gyrA462 via electroporation. Do Mini-Prep and screen correct plasmid by restriction analysis
construct the plasmid pBBR1-kan-cm-promoter(1-25)-firefly via liner circular recombination in GB05 red gyrA462. Do Mini-Prep and screen correct recombinants by restriction analysis.
transform the plasmid pBBR1-kan-amp-ccdb-BAD-GFP-firefly into p.fluorescence pf-5 via electroporation.
Chracterize the strength of different promoters by luciferase firefly assay.
Colleborate with seven Chinese iGEM teams to characterize the promoter strength under differdent conditions.
modeling
constract our software tool