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Team:St Andrews/Procedure
From 2012.igem.org
Lab Book
Procedure
Sequences Primers Sequencing results Lipid analysis Mass Spectrometry
Start from lipid extraction
primers, sequence results
Sequences
Δ12 T. Cruzi
ATGTCGCGGGTTGACAATTTAACTGTCGCGCCGGGTCCACCGGATGTCATGAAGGCGGTG CTTAAGTCTGGGCGCAACAAGGAGCACAATGTCATTGTGCCTTCCTCCGCACTGACCATTG GAGAAATTCAGGAGAAAATCCCGGTAAAGTACTTTGAACGCAACACTACCCGGAGCGTCA TGTTTCTTTTACGCGACCTCGCACAGGTGGCGTTGACGTACGCCATTATGTACGCCGTCGCC TTGCCACTTGCCACTTCCATGGAAGTTTCTGCAGCAAGAACGTTGGCCGACGGTGGGGTGT TATCATTGATGGCAGGTACAGCCATGACGACAGCGGCGTGGCTTTTAAAGGCAGTGTTGTG GGCGGTGTTTTGGTTTGTACAGGGACTTAACGGCACTGCGCTTTGGGTCCTGGCTCACGAA TGCGGCCATCAGGCGTTCAGCCCCATGAAAGGTTTGAATGACGCCGTTGGCATGATTTTGC ACTCTGCGCTGCTCGTGCCGTACCACAGCTGGCGCATCACCCACGGCGGCCACCACAAACA CACGAATCACCTCACGAAGGATCTTGTGTTTGTTCCAGACAAACGAAACGCGGTTGTGGAG CTCGTGGAGGAGGCGCCGCTGGTGTTATTAATTCAATTATTGCTGATTTTTCTCTTTGGTTG GCCGGCACATCTTCTTTTTAATGCCTCTGGACAGGAATTTGGCCGACTCGCGAGCCACTTTG ACCCCGGCGCTCCATTTTTCCGCAGCGAAGACCGTCACGATATTGTCCTGTCGAATGTTGGG ATTGTCAGCGCGTTATTTGTCATTTTCTCCAGCGTTTACCGCTTTGGTTTTACAAATGTTTTCT GCTGGTACATTGTACCGTACCTCTGGGTGAACTTTTGGCTG TGTACATTACATACCTGCAGCACACGGATATACGCATTCCTCACTACACACATGAGCACT GGACGTTTGTTCGCGGTGCATTGGCGGCTGTGGACAGGGACTACGGCTTTGTCCTTAACA CATGGCTCCATCACATCAATGATTCCCACGTGGTACATCACCTCTTTAGCAAAATGCCAC ACTACAACGCAATCCAGGTGACAAGAAAGTACATTCGTGAGATACTGGGTGCCACATACA TTACGGATGAGAGGTCACTGTGGAAGATGCTCTGGGAACAGCGTAGAGAGTGCCGCTATG TTGTTCCCGCAGAGGGCGTCTGTGTCTTTCATGGGTAA
Δ12 Synechocystis
ATGACTGCCACGATTCCCCCGTTGACACCAACGGTAACGCCCAGCAATCCCGATCGCCCG ATTGCGGATCTCAAACTACAAGACATCATTAAAACCCTGCCCAAGGAATGCTTCGAGAAA AAAGCGAGCAAAGCCTGGGCTTCTGTTTTGATTACCCTAGGGGCGATCGCCGTGGGCTAT TTGGGCATTATTTATCTGCCCTGGTACTGCTTGCCCATTACCTGGATCTGGACAGGGACA GCCTTAACGGGGGCCTTCGTTGTCGGCCATGACTGTGGCCATCGCTCCTTTGCTAAAAAA CGCTGGGTCAATGATTTAGTGGGACATATCGCTTTTGCTCCCCTCATCTACCCTTTCCAT AGCTGGCGCCTACTCCACGACCACCATCACCTCCACACCAACAAAATTGAGGTTGATAAC GCCTGGGATCCCTGGAGTGTGGAAGCTTTCCAAGCCAGCCCGGCGATCGTCCGGCTTTTT TATCGGGCCATCCGGGGTCCCTTCTGGTGGACTGGTTCCATTTTCCATTGGAGCTTAATG CACTTCAAACTTTCCAACTTTGCCCAAAGGGACCGCAATAAAGTCAAATTATCCATTGCC GTTGTCTTCCTGTTTGCGGCGATCGCCTTTCCTGCCCTAATTATCACCACAGGGGTGTGG GGTTTCGTCAAATTTTGGCTAATGCCCTGGTTGGTGTATCACTTTTGGATGAGCACTTTT ACCATTGTGCACCACACCATTCCCGAAATTCGTTTCCGTCCCGCCGCCGATTGGAGTGCC GCCGAAGCCCAGTTAAATGGTACTGTTCACTGCGATTATCCCCGTTGGGTGGAAGTGCTC TGCCATGACATCAACGTCCATATTCCCCACCACCTCTCCGTTGCCATCCCTTCCTATAAC CTACGACTAGCCCACGGAAGTTTAAAAGAAAACTGGGGACCTTTTCTTTACGAGCGCACC TTTAACTGGCAATTAATGCAACAAATTAGTGGGCAATGTCATTTATATGACCCCGAACAT GGCTACCGCACCTTCGGCTCCCTGAAAAAAGTTTAA
Δ15 Synechocystis
GTGCGTCTAGAAATTTCATCGCCTCAAACAAAGCTTCCTTACCCCAAAACTGAAGAATTA CCATTTACCCTCCAAGAGCTCAGAAACGCTATTCCAGCGGATTGTTTTGAGCCATCGGTA GTCCGGTCCTTGGGCTACTTTTTTTTGGATGTTGGTTTAATTGCCGGGTTTTATGCTCTA GCGGCCTACCTTGATTCCTGGTTCTTCTATCCGATTTTTTGGTTAATTCAGGGAACCCTA TTCTGGTCCCTGTTTGTGGTGGGCCATGATTGTGGCCATGGCTCCTTTTCCAAATCCAAA ACCCTTAATAATTGGATTGGTCATCTCAGCCACACGCCAATTTTGGTGCCTTACCATGGC TGGCGTATTAGTCATCGTACTCACCATGCCAACACGGGCAATATCGACACCGACGAAAGT TGGTATCCAGTGTCGGAGCAAAAATATAACCAAATGGCCTGGTATGAAAAACTTCTACGT TTTTACTTGCCTCTGATCGCCTACCCCATTTATCTATTTCGGCGATCGCCAAACCGGCAA GGCTCCCATTTCATGCCCGGCAGTCCCCTATTCCGTCCCGGAGAAAAAGCAGCTGTTCTC ACCAGCACCTTTGCCCTTGCAGCCTTTGTCGGCTTCCTTGGCTTTTTAACTTGGCAATTT GGCTGGCTATTTTTGCTGAAATTTTATGTTGCCCCCTACCTCGTGTTTGTGGTGTGGTTA GATTTGGTCACATTTTTACATCACACTGAAGACAATATCCCTTGGTATCGTGGTGATGAC TGGTATTTTCTCAAAGGTGCCCTCTCCACCATTGATCGGGATTACGGCTTCATTAACCCC ATTCACCATGACATTGGCACCCACGTCGCCCACCATATTTTCTCGAATATGCCCCACTAC AAGTTACGCCGGGCGACTGAAGCCATCAAGCCCATTTTAGGGGAATATTATCGATATTCT GACGAGCCAATTTGGCAAGCTTTTTTTAAGTCCTACTGGGCTTGCCATTTTGTTCCTAAT CAAGGTTCAGGGGTCTATTACCAATCCCCATCCAATGGTGGATATCAAAAGAAACCTTAA
Δ6 Synechocystis
ATGCTAACAGCGGAAAGAATTAAATTTACCCAGAAACGGGGGTTTCGTCGGGTACTAAAC CAACGGGTGGATGCCTACTTTGCCGAGCATGGCCTGACCCAAAGGGATAATCCCTCCATG TATCTGAAAACCCTGATTATTGTGCTCTGGTTGTTTTCCGCTTGGGCCTTTGTGCTTTTT GCTCCAGTTATTTTTCCGGTGCGCCTACTGGGTTGTATGGTTTTGGCGATCGCCTTGGCG GCCTTTTCCTTCAATGTCGGCCACGATGCCAACCACAATGCCTATTCCTCCAATCCCCAC ATCAACCGGGTTCTGGGCATGACCTACGATTTTGTCGGGTTATCTAGTTTTCTTTGGCGC TATCGCCACAACTATTTGCACCACACCTACACCAATATTCTTGGCCATGACGTGGAAATC CATGGAGATGGCGCAGTACGTATGAGTCCTGAACAAGAACATGTTGGTATTTATCGTTTC CAGCAATTTTATATTTGGGGTTTATATCTTTTCATTCCCTTTTATTGGTTTCTCTACGAT GTCTACCTAGTGCTTAATAAAGGCAAATATCACGACCATAAAATTCCTCCTTTCCAGCCC CTAGAATTAGCTAGTTTGCTAGGGATTAAGCTATTATGGCTCGGCTACGTTTTCGGCTTA CCTCTGGCTCTGGGCTTTTCCATTCCTGAAGTATTAATTGGTGCTTCGGTAACCTATATG ACCTATGGCATCGTGGTTTGCACCATCTTTATGCTGGCCCATGTGTTGGAATCAACTGAA TTTCTCACCCCCGATGGTGAATCCGGTGCCATTGATGACGAGTGGGCTATTTGCCAAATT CGTACCACGGCCAATTTTGCCACCAATAATCCCTTTTGGAACTGGTTTTGTGGCGGTTTA AATCACCAAGTTACCCACCATCTTTTCCCCAATATTTGTCATATTCACTATCCCCAATTG GAAAATATTATTAAGGATGTTTGCCAAGAGTTTGGTGTGGAATATAAAGTTTATCCCACC TTCAAAGCGGCGATCGCCTCTAACTATCGCTGGCTAGAGGCCATGGGCAAAGCATCGTGA
ELO 6 L. major
ATGAAGGTCATCGTCGCTTCTGGCCCGGACGGTGCCCGCAAGCACGAGGTGGAGCTGGCA GCCAACGCCACGCTCGCAGATCTGAAGAAGGCCTACCAACGGGGTGTGGACGTGCACCGC AAGTCGTTCAAGGTTCCCAGCGCGGAGTCGCCGCTGCCAGGTGCGGATAGTGGCAAGCTG CGCCCGAACCTCATTACTCTGTCAGATAAGGTGCCCCTGTCGCAGCAGGGGGTGAAGGAT GGCTCGGTGATCACTTACAAGGACCTCGGCCCGCAGATCGGCTACCGCACGGTGTTCTAC GTCGAGTATGCCGGCCCCATCGCCTTCATGCTGCTGTACGCCATGCGCCCTTCGCTCATC TACGGCTCTGCCCCGATGCCGGCTTACGGCTACACGCAGAAGCTATACATTGGCCTCTTC CTCGCCCACTTCTTAAAGCGCGAGCTCGAGTCCATGTTTGTGCACAAGTTCTCGCACCCA ACGATGCCGATGCGCAACATCTTCAAGAACTGCATCTACTACTGGTCCTTCGCCGCCTTC ATCGGCTACGTGCTGTGCAGTCCTTCATTCACGCCGACCAGCACCATGCAGTCAAACTTC GGCGCCGTGGTCATGGTCATCAACGAGCTGCTGAACTTCGCGGTGCACTACCAGCTTAGC ACGATGCGCAAGTCCGATGGTGACACCACCCGCAACGTGCCGCAAGGCCCTCTGTTCGCC TTCGTCTCGTGCCCGAACTACTTCTTTGAGATTATGTCGTGGGTGTCCTTTTCCATCGGC ACAAATATGTTATCCTCCTGGTTCTTCACACTCGCCGGTTTCGTGCAGATGGCGGACTGG GCGAAGAAGAAGCACCGGGGCTACGTCAAGGCGGACCCGGCCAATAAAAAGAAGGCCGCC ATTCTGCCCTTCATCATGTAG
Primers
All primers are notated 5' to 3'.
When using the pET-duet vector, we needed additionally primers for the alternative restriction sites and His-tags.
Sequencing results
- 16/7/12 Δ15
- Δ15 and Δ6 desaturases from Synechocystis in pET-15b were sent off for sequencing. Once again, the results were discouraging even though protein and lipid analysis showed the opposite. BLAST showed that the gene found in both samples corresponded to a T. brucei HMG-CoA reductase which we thought we had cut out from the plasmid. We thus performed a PCR, using the vectors sent off for sequencing as a template.
- Sequence
Δ15 Syn. in pET-15b
atATACCAtggGcAgCAGccaTcnnCATcaTCAtnncAGCagCGGCCTggtgCCGCGCGGCAGCCATATGCTTCgtagga cTCTCTCatgtgCTtgtGGGACAGGCAAAACGGGttgGGGCGCAAtgaGCAATGCCgagCTTGTTaAAGCcgtAAGCAgt cGTCAAttAACCTTTTATGGGTTAGAGCacgCTCTGgagCCCGACTACCAGCGCGCTATCGcTgtGCGTCGtgAGGTGgt gtCCGACTAtgtgGCATCCTCCCAAAgTGCggaGGTGAAGAGGAAAAGCTTGGAGCGTATACCATTTGAAAACTACGAAT GGGATCGAGTGGTTGGTCAGAACTGTGAAAATATTGTGGGTTACGTTCCTATTCCTCTTGGTATGGCTGGGCCAATAATA ATGGATGGCTGTGAATACCCCATTCCCATGGCCACGACAGAAGGTGCCCTggtTGCCAGCACACATCGAGGCGCACGAGC TATATCCCAAAGTGGGGGCTGTAAGACACTGATCTTGGGCGAGGGCATGTCACGTGCTCCAGTAGTGGAGGTTGAGTCTT TGGAAGAGGCGGGAAggcttCATAATTTTTGTGTagaAAAtttcaCTGAAATCAAAGCAGCTTTCGAGagaCGACGCGAT TTGGGAattGCAGTCGCTCAAGTGCGTCATCATCGGTCnnnannCCTATATTCggTTCCGCGCGAccaCCGGagatGCga tg
- 16/7/12 Δ6
- Sequence
Δ6 Syn. in pET-15b
AnTTCCCCcTctaGaaTaTTTTTGTTtACTTtAAGaAGGAGATATACCATGGGCAGCAGCCATcncTcAtcaTCATCACA GCAGCGGCCTGGTGCCGCGCGGCAGCCATATGCTTCGTAGGACTCTCTCATgtGCTTGTGGGACAGGCAAAACGGGTTGG GGCGCAATGAGCAATGCCGaGCTtgTTAAAGCCGTAAGCAGTCGTCAATTAACCTTTTATGGGTTAGAGCAGGCTCTGGA GCCCGACTACCAGCGCGCTATCGCTGTGCGTCGTGAGGTGGTGTCCGACTATGTGGCATCCTCCCAAAGTGCGGAGGTGA AGAGGAAAAGCTTGGAGCGTATACCATTTGAAAACTACGAATGGGATCGAGTGGTTGGTCAGAACTGTGAAAATATTGTG GGTTACGTTCCTATTCCTCTTGGTATGGCTGGGCCAATAATAATGGATGGCTGTGAATACCCCATTCCCATGGCCACGAC AGAAGGTGCCCTGGTTGCCAGCACACATCGAGGCGCACGAGCTATATCCCAAAGTGGGGGCTGTAAGACACTGATCTTGG GCGAGGGCATGTCACGTGCTCCAGTAGTGGAGGTTGAGTCTTTGGAAGAGGCGGGAAGGCTTCATAATTTTTGTGTAGAA AATTTCACTGAAATCAAAGCAGCTTTCGAGAGCACGACGCGATTTGGGAAGTTGCAGTCGCTCAAGTGCGTCATCATCGG TCGCAnAGCCTATATTCGGTTCCGCGCGACCACCGGAGATGCGATGGGGATGAACATGATAACCAAAGGTGTCGACAAGG CATTGGAGTTAAtanAGCAnAACTTTCCGTCGATGAAGGTTATTGCGTTGTCGGGGAACTACTGCACTGanaagAAACcC TCCGCAGTGAACTGGATTGaggggaaGAGGAAnAAaCTGTcnnttgCCgaagGCGCTcgtaaaggggCgaAGt
- 29/6/12 ELO6
- After showing successful insertion of L. major Δ6 elongase in pET-15b, the sample was sent off for sequencing. Unfortunately, the gene sequenced was not the Δ6 elongase we were hoping to find, but a T. brucei mevalonate kinase, which was previously inserted in the pET-15b vectors we had been using. Thus, we digested some fresh pET-15b to be used in the future.
- Sequence
ELO6 in pET-15b
GnanTTTGTTtAcTttaagaaggagaTATACCATGGGcAGCAGCCATcatCATcatcaTCACAGCAGCGGCCTggtGCCG CGCGGCagCCATATGGTCGTGGCATCGtgTCCGGGAAAAGTGCTGATTCTTGGTGGTTAcCTAATAGttGAGGAGCCAAA TGTTGGTATTTCCGTAGGAACAACCGCGCGCTTCGTGACACGTGTGGCGTCgtgGAAAAAATGCTCTGATGGAAAGTGTC GAGTGCACATCGTCTCGTCACAGTTCAACAAggaGTTTACGTTTGAATGCGCGGCTGAAGAGGACAGTGATTCCACTATC AAGATTGTGCAATTAGAGGGTGCACCATCGCCGTTTCTCTTCTACGGTATTTTGTACTCGGTTGCCGGCGCTCTATTATT TGGTGGTGATATTTTTCGTGATGTTACCCTAGAGTTGTTGGCGGATAACGATTTCTACAGCCAAAGGAATTATTTGGAGT CCCAAGGCAAGCCGGTGACCGCAGCCAACCTGCGGCTCATCCCCCGTTATACGCCCCTCTTGGGGGAGGTTTCCAAAACT GGTCTAGGATCTTCAGCGGCAATGACCACTAGTGTCGTAGCGTGCCTGCTCCAACTTTTTGTTTTTGATTCGAAGAAAAA CAATGCCACTGAGTCCGTGGAGAGGGCACCGGAGCTACCTCTACGTTTGGAGGATGTAACAGAGTTCATTCATCGTATTA GTCAGGTTGCCCACTGCGTAGCACAGGGAAAAGTGgGGAGCGGGTTTGATGTCTACACGGCCACGTTCGGGACGTGTGTG TACCGACGGTTCTCCGCACGCGTGCTGGAnAAGCTCGTAAAGGgtAATGAACCTCCGAAGCGCGTCGCTATTCCTCTTTT ACGTGAGTGTGTCGAGACGGATGAAGTGTGgGTTCAGAGGATACcCTtccGGCTTCCTACCGGGTTGCAGCTTCTTTTAG GGGATGTACACAAGGGAGgAacaGAAAcACCTGGCATGGTGTcgaAGGTCATGTCatgnnagnggTCGGTGACCacGGAC CCcAACAGnttgnggnnnngantTcCCATgnntAAT
- 23/7/12 Δ12
- 15b forward gave 0 nucleotides
- 15b reverse gave 0 nucleotides
- 20b forward gave a 41 nucleotide result.
SequencegCGnCctGntGCCgCGCGGCnnnnnnnnnnnnnnnnnngac- A BLAST search showed this to be compatible with Crocosphaera watsonii (genome shotgun sequence, 21%), a diazotrophic cyanobacteria.
- 20b reverse gave 0 nucleotides
Lipid analysis
Lipids were extracted from transformed E. coli using the Blight/Dyer method. (Please refer to the Lipid extraction lab book entry for further detail.)
We then analyzed the the fatty acid content of the transformed cells to determine whether it differed from the previously analyzed background lipid composition. This was done using mass spectrometry.
Mass SpectrometryPrimers Digestion Transformation
Primers
All primers are notated 5' to 3'.
Please refer to the Primer annealing lab book entry for further detail.
For primer annealing in the PCR, the primer sequences were combined in the following way:
Please refer to the Primer annealing lab book entry for further detail.
Digestion
pGEX-6P-1 was cut using EcoR1(954) and Xho1 (975)
Transformation
pGEX vector with insert transformed into DH5α E. coli cells to replicate.
pGEX vector with G-Block insert transformed into DH5α E. coli cells to replicate.
pGEX vector with insert transformed into BL21 E. coli cells for protein expression.
pGEX vector with G-Block insert transformed into BL21 E. coli cells for protein expression.
