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Team:St Andrews/Procedure
From 2012.igem.org
Lab Book
Procedure
- date
- what we did
- date
- what we did
- date
- what we did
anyone really keen to write this up, up to protein visualization? Don't forget to mention the differing restriction enzymes and vectors of each team. I can add in links to the protocols page.
Please also refer to our Protocols page.
Sequences Primers Sequencing results Lipid analysis
Start from lipid extraction
primers, sequence results
Sequences
Δ12 T. Cruzi
atgtcgcgggttgacaatttaactgtcgcgccgggtccaccggatgtcatgaaggcggtgcttaagtctgggcgcaacaaggagcacaat gtcattgtgccttcctccgcactgaccattggagaaattcaggagaaaatcccggtaaagtactttgaacgcaacactacccggagcgtc atgtttcttttacgcgacctcgcacaggtggcgttgacgtacgccattatgtacgccgtcgccttgccacttgccacttccatggaagtttctg cagcaagaacgttggccgacggtggggtgttatcattgatggcaggtacagccatgacgacagcggcgtggcttttaaaggcagtgttgtg ggcggtgttttggtttgtacagggacttaacggcactgcgctttgggtcctggctcacgaatgcggccatcaggcgttcagccccatgaaag gtttgaatgacgccgttggcatgattttgcactctgcgctgctcgtgccgtaccacagctggcgcatcacccacggcggccaccacaaac acacgaatcacctcacgaaggatcttgtgtttgttccagacaaacgaaacgcggttgtggagctcgtggaggaggcgccgctggtgttatt aattcaattattgctgatttttctctttggttggccggcacatcttctttttaatgcctctggacaggaatttggccgactcgcgagccactttgac cccggcgctccatttttccgcagcgaagaccgtcacgatattgtcctgtcgaatgttgggattgtcagcgcgttatttgtcattttctccagcgt ttaccgctttggttttacaaatgttttctgctggtacattgtaccgtacctctgggtgaacttttggcttgtgtacattacatacctgcagcacacg gatatacgcattcctcactacacacatgagcactggacgtttgttcgcggtgcattggcggctgtggacagggactacggctttgtccttaac acatggctccatcacatcaatgattcccacgtggtacatcacctctttagcaaaatgccacactacaacgcaatccaggtgacaagaaag tacattcgtgagatactgggtgccacatacattacggatgagaggtcactgtggaagatgctctgggaacagcgtagagagtgccgctatg 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
- date and gene
- result
- date and gene
- result
- date and gene
- result
- 23/7/12 Δ12 Syn. in pET-15b and 20b
- 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. Add in relations to synechocystis.
- 20b reverse gave 0 nucleotides
Lipid analysis
who knows what those Metal Mickies are up to. Eating nachos in the Whey Pat most likely. MEGA LOLS (all caps, with a Z).
PrimersDigestionTransformation
Primers
All primers are notated 5' to 3'.
For primer annealing in the PCR, the primer sequences were combined in the following way:
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.
