Difference between revisions of "Part:BBa K638402"
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<partinfo>BBa_K638402 parameters</partinfo>
 
<partinfo>BBa_K638402 parameters</partinfo>
 
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===Producing BBa_k638402 from primer synthesis===
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We assembled the basic part by using a pair of primers that will anneal to give the whole sequence. Biobrick prefixes and suffixes can then be added to create an in-frame fusion by assembly techniques such as [https://parts.igem.org/Plasmid_backbones/Assembly_of_protein_fusions BBF RFC 23 or 25].  Alternatively, Cambridge 2011 created scar-free fusions of this export tag to our protein of interest by [http://www.cambridgeigem.org/RFC57.pdf Gibson Assembly].
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{| border="1" align="center" style="text-align:center;"
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!Name
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!Sequence
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!Tm
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|-
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|TorA FWD
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|ATGGCGAACAACGACTTATTTCAGGCTTCTCGGCGTCGCTTTCTGGCGCAGCTGGGCGGATTAACGGTGGCGGGT
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|70.98&deg;C
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|-
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|TorA REV
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|TGCGGCTTGTGCTGCCGTCGCTCTGCGAGGAGTCAACAGCGACGGGCCCAACATACCCGCCACCGTTAATCCGCC
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|70.98&deg;C
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|}
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Thermocycler profile: 10 cycles:
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Melt 95&deg;C 10 sec /
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Anneal 65&deg;C 30 sec /
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Extend 72&deg;C 20 sec

Revision as of 11:09, 21 September 2011

TorA tag variant

This is an improved version of part BBa_K233307 designed to allow comparison with measurements of functionality in the literature, and to make it easier to synthesise.

This TorA leader sequence variant has had been successfully used to export GFP to the periplasm of E.coli as described [http://www.ncbi.nlm.nih.gov/pubmed/11123687 here].

We ordered this sequence as a synthetic oligonucleotide in two halves: one 5-->3, one 3-->5, with 20bp overlap. We then joined these parts using PCR to make the full sequence. This is essentially a deliberate primer dimer. We inserted this sequence into our previous constructs using [http://www.synbio.org.uk/gibson/downloads/files/RFC57.pdf Gibson assembly], an extremely powerful method of assembling DNA constructs that is compatible with Standard Assembly.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Producing BBa_k638402 from primer synthesis

We assembled the basic part by using a pair of primers that will anneal to give the whole sequence. Biobrick prefixes and suffixes can then be added to create an in-frame fusion by assembly techniques such as BBF RFC 23 or 25. Alternatively, Cambridge 2011 created scar-free fusions of this export tag to our protein of interest by [http://www.cambridgeigem.org/RFC57.pdf Gibson Assembly].

Name Sequence Tm
TorA FWD ATGGCGAACAACGACTTATTTCAGGCTTCTCGGCGTCGCTTTCTGGCGCAGCTGGGCGGATTAACGGTGGCGGGT 70.98°C
TorA REV TGCGGCTTGTGCTGCCGTCGCTCTGCGAGGAGTCAACAGCGACGGGCCCAACATACCCGCCACCGTTAATCCGCC 70.98°C


Thermocycler profile: 10 cycles: Melt 95°C 10 sec / Anneal 65°C 30 sec / Extend 72°C 20 sec