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Team:St Andrews/project/laboratory
Laboratory
Contents |
Introduction
The synthetic biology portion of our project has provided the undergraduate members of our team with a rare opportunity to work in the lab for an extended period of time. Many of us have gained important skills such as learning to use our time efficiently, planning the use of limited materials and organising experiments effectively.
Overview
The lab work has focused on constructing two main biobricks - the bistable switch and the CAI-1 sender.
The bistable switch
The CAI-1 sender
This biobrick is essential to our project. CAI-1, the cholera-specific auto inducing molecule, can switch off the production of cholera toxin by V. cholerae when it is present in high concentrations. To make this biobrick the gene responsible for making the CAI-1 synthase (cqsa) was optimised for E. coli and then synthesised by geneart. CQSA can be found [http://partsregistry.org/Part:BBa_K356001 here].
first we found the cholera gene for Vibrio Cholera autoinducer synthase which is as follows.
>vch:VCA0523 hypothetical protein; K10915 CAI-1 autoinducer synthase [EC:2.3.-.-] (N)
atgaacaagcctcaacttcctgattttattcagaacaagatagatcactatattgaaaat
tattttgatataaacaaaaacggtaaacaccttgtattgggtaaacaagccagccctgat
gacattattttgcaaagtaatgattatctcgcattggccaatcatccgttgatcaaagct
cgtttggcgaagtcattactggaagaacaacaaagcttatttatgtcagcctcatttcta
caaaatgactatgacaaacccatgattgagaaacgtctggctaagttcacaggctttgat
gaatgtctattatctcaatctggttggaatgcaaacgtcggtttattacaaaccatctgc
cagcccaatacgaatgtttacattgattttttcgcgcacatgtcgttatgggaaggggcg
cgctacgccaatgctcaggcgcatccttttatgcataataactgtgaccatttacgtatg
ctgattcaacgtcatggtcctgggatcattgtcgtagactcgatttacagcactttaggt
acgattgcaccgctagcggaactggtcaatatcagtaaagagtttggctgcgccttatta
gtcgatgaatcccactctttgggcacacatggccctaatggtgcaggtttattggcagaa
ttaggcctcactcgtgaagtgcattttatgaccgcaagtttggccaaaacctttgcttat
cgcgcaggagccatttggtgtaacaatgaagtgaatcgctgcgttccttttattagttat
ccagctatttttagttctactttgctgccttatgaagcggcaggattagaaacgacttta
gagattattgaatctgcggataatcgtcgtcagcatttagatcgtatggcaagaaaatta
cgcataggattatcccagctgggattaaccattcgcagtgaaagccaaattattggtcta
gaaacaggagatgaacgaaataccgaaaaagttcgggattatttagaaagtaatggagtg
tttggctcagtattctgccgcccggcaacttcaaagaataaaaacattattcgcttatca
ctcaatagtgatgtgaacgatgagcaaatcgccaaaataattgaggtttgctctgatgcg
gtcaactacggtgatttttattttcgttaa
[http://www.genome.jp/dbget-bin/www_bget?vch:VCA0523 the CAI-1 autoinducer synthase at genome.jp]
we then codon optomised this gene for E.coli using the functionality built into [http://mrgene.com/desktopdefault.aspx/tabid-2/ the Mr Gene Page]
which gave us
ATGAACAAACCTCAGCTGCCTGACTTTATCCAAAACAAAATCGACCACTATATCGAGAACT
ATTTCGACATTAACAAAAACGGCAAACACCTGGTGCTGGGCAAACAAGCATCACCGGATGA
CATTATCCTGCAAAGCAACGACTATCTGGCCCTGGCTAATCACCCTCTGATCAAAGCTCGT
CTGGCGAAAAGCCTGCTGGAAGAACAGCAATCCCTGTTTATGAGCGCCTCCTTTCTGCAAA
ACGATTATGACAAACCGATGATTGAGAAACGCCTGGCCAAATTCACTGGTTTCGATGAATG
CCTGCTGTCTCAGTCTGGTTGGAATGCCAATGTTGGTCTGCTGCAAACAATCTGTCAGCCT
AACACGAACGTCTATATTGATTTCTTCGCCCACATGTCGCTGTGGGAAGGTGCTCGTTATG
CTAATGCTCAGGCCCATCCGTTTATGCACAACAACTGTGACCATCTGCGTATGCTGATTCA
GCGTCACGGTCCTGGTATTATCGTCGTGGACTCCATCTATTCTACCCTGGGGACCATTGCT
CCACTGGCTGAACTGGTGAATATCAGTAAAGAGTTTGGGTGTGCCCTGCTGGTTGATGAAA
GCCATTCTCTGGGAACCCATGGCCCGAACGGTGCCGGGCTGCTGGCGGAGCTGGGTCTGAC
ACGTGAAGTTCACTTCATGACCGCTTCGCTGGCAAAAACATTCGCCTATCGTGCTGGTGCC
ATTTGGTGTAACAACGAGGTTAATCGCTGTGTTCCGTTCATCTCTTATCCGGCCATCTTTA
GCAGTACACTGCTGCCGTATGAAGCTGCTGGTCTGGAAACAACCCTGGAGATTATCGAGTC
TGCCGATAACCGTCGTCAACATCTGGATCGTATGGCCCGTAAACTGCGTATTGGTCTGTCC
CAACTGGGTCTGACAATTCGTAGCGAATCTCAGATTATTGGCCTGGAGACTGGTGACGAGC
GTAATACCGAGAAAGTCCGTGATTATCTGGAGTCTAACGGCGTGTTTGGTAGCGTTTTTTG
TCGTCCGGCAACCTCTAAAAACAAAAACATCATCCGCCTGTCCCTGAATAGTGATGTGAAT
GATGAGCAGATCGCTAAAATCATTGAAGTCTGTTCGGATGCCGTGAATTATGGTGACTTCT
ATTTCCGCTGA
We then added the biobrick [http://partsregistry.org/Help:BioBrick_Prefix_and_Suffix prefix], promoter [http://partsregistry.org/Part:BBa_J23100 J23100], ribosome binding site [http://partsregistry.org/Part:BBa_B0034 B0034] before the CQSA sequence
and the terminator [http://partsregistry.org/Part:BBa_B0015 B0015] and the biobrick [http://partsregistry.org/Help:BioBrick_Prefix_and_Suffix suffix], after the sequence. We then sent these details of to Geneart
and got them to do the hard work of synthesising our part for us.
once we received our DNA from synthesis we transformed it into E.coli and grew up some colonies.
Unfortunately it is not currently possible to get parts synthesied and sent ready in the biobrick
so we made an over night broth culture of a selection of colonies and then miniprepped them the
following morning. We digested the resulting DNA with the restriction enzymes EcoR1 and Spe1 and
ran a gel to ensure that we had the part sizes that we wanted, we got further confirmation using
colony PCR with the [http://partsregistry.org/Part:BBa_G00100 VF2],[http://partsregistry.org/Part:BBa_G00101 VR] primer pair
we used gel extraction to get the parts of the size we wanted and ligated them into [http://www.example.com psb1c3]
we did more confirmation using digestion+gel and colony PCR and sent our favourites to MIT
As you might imagine this is a very simplified account, for the true horror of our summer see below.
