Team:St Andrews/Procedure

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Revision as of 09:48, 27 July 2012

St Andrews iGEM '12

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'.
 

Δ12 T. Cruzi forward
    ATATCATATGTGGCGGGTTGACAATTTA
Δ12 T. Cruzi reverse

    ATATCTCGAGTTACCCATGAAAGACAC

Δ12 Synechocystis forward
    ATATCATATGACTGCCACGATTCCC
Δ12 Synechocystis reverse
    ATATCTCGAGTTAAACTTTTTTCAGGGAGC
Δ15 Synechocystis forward
    ATATCATATCGCTCTAGAAATTTCATC

Δ15 Synechocystis reverse
    ATATCACGACTTAAGGTTTCTTTTGATATC
Δ6 Synechocystis forward
    ATATCATATGCAAACAGCGGAAAGAATT
Δ6 Synechocystis reverse
    ATATCTCGAGTAACGATGCTTTGACCATGGCCTCTAG


 

When using the pET-duet vector, we needed additionally primers for the alternative restriction sites and His-tags.

ELO6 L. major forward
    TATCATATGGGAAGCAGCCATCATCATCATCATCACAGCATGGTCTCTCTGGAGCAGGCC
Δ15 T. Cruzi forward
    TATCATATGGGCAGCCATCATCATCATCATCACAGCATGCGTCTAGAAATTTCATCG
Δ6 T. Cruzi forward
    GGATCCGAATTCATCTAACAGCGGAAAGAATT
Δ6 T. Cruzi reverse
    GACAAGCTTTCACGATGCTTTGCCCATGGCCTCTAC
Δ12 T. Cruzi forward
    GGATCCGAATTCATGACTGCCACGATTCCC
Δ12 T. Cruzi reverse
    GACAAGCTTTTAAACTTTTTTCAGGGAGC

 
Sequencing results
 
date and gene
result
date and gene
result
date and gene
result
23/7/12 Δ12 Syn.

15b forward gave 0 nucleotides

15b reverse

20b forward gave a 41 nucleotide result.

Sequence
    gCGnCctGntGCCgCGCGGCnnnnnnnnnnnnnnnnnngac

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

Launch Modal

 

 
Lipid analysis
 

 

 

 

 

 

 

 

 

who knows what those Metal Mickies are up to. Eating nachos in the Whey Pat most likely.

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University of St Andrews, 2012.

Contact us: igem2012@st-andrews.ac.uk, Twitter, Facebook

This iGEM team has been funded by the MSD Scottish Life Sciences Fund. The opinions expressed by this iGEM team are those of the team members and do not necessarily represent those of Merck Sharp & Dohme Limited, nor its Affiliates.

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