Generator

Part:BBa_K1807002:Design

Designed by: Ivan Gyulev   Group: iGEM15_York   (2015-09-17)
Revision as of 00:04, 19 September 2015 by Isgyulev (Talk | contribs) (→‎Source)

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Escherichia coli Polyphosphate Kinase Enzyme


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 396
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1750
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This protein generator device contains the Escherichia coli Polyphosphate Kinase 1 (PPK1) enzyme and has had its illegal Restriction sites removed during assembly.

The part was generated using the following primer pairs:

Fragment 1

Forward: 5' AGCACATACGAGAAAGAGGAGAAATACCCCATGGGTCAGGAAAAGCTATACATCGAAAAA 3'

Reverse: 5' TAGAGGCCGTCGAAcTCCTGATCGGCTTTC 3'

Fragment 2

Forward: 5' GAAAGCCGATCAGGAgTTcGACGGCCTCTA 3'

Reverse: 5' GAGCCTTTCGTTTTATTTGATGCCTGGCCCTTATTCAGGTTGTTCGAGTGATTTGATGT 3'


Fragment 1's For and Fragment 2's Rev Primers contain a point mutation that was designed to remove the illegal EcoRI restriction site. Those two primer pairs are overlapping. The two fragments were produced in independent PCR reactions and were then mixed together with SmaI-linearized vector BBa_K1807000 in a Gibson Assembly Reaction to generate BBa_K1807002.

Source

Escherichia coli W3110

References