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Part:BBa_K1122674

Designed by: Aleksandra Lewicka, Jan Lyczakowski   Group: iGEM13_Edinburgh   (2013-08-22)
Revision as of 13:52, 3 October 2013 by JanL (Talk | contribs) (Generation of protein fusion)

Plac+fused PDC-ADH

lac promoter plus PDC-ADH fusion protein for ethanol production


Generation of protein fusion

In order to generate fusion of pyruvate decarboxylase (pdc) and alcohol dehydrogense B (adhB) Mutagenesis with Blunt- Ended Ligation (MABEL) was used.

A pair of primers was designed complementary with 3' end of pdc and 5' end of adhB. Figure 1 represents the MABEL process.


Bioethanol1.jpg

Fig1. Represents MABEL process used for generation of fused pdc-adhB construct. Primer sequences used: Forward: GCATCAAGCACCTTTTATATCC; Reverse: CAGCAGTTTATTCACCGGTTTAC. See appendix of the Edinburgh University 2013 iGEM team figure 1 for full details on the part sequence, primer binding sites and deleted region.


Generated PCR product (see Fig 1.) was analysed on an agarose gel (Fig 2.):

Bioethanol2.jpg

Fig 2.Presence of a single PCR product of correct size (app. 6000 bp) on a 0.8% agarose gel. 1kb NEB DNA ladder was used. Several replicates of the reaction were loaded on a gel.

Evidence for presence of protein fusion

DNA level evidence


Bioethanol3.jpg

File:Bioethanol sequencing.zip


Protein level evidence


Bioethanol4.jpg

Bioethanol5.jpg

Bioethanol6.jpg


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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1109
    Illegal AgeI site found at 2315
  • 1000
    COMPATIBLE WITH RFC[1000]

Functional Parameters

Bioethanol7.jpg

Activity of adhB as a fusion member


Bioethanol8.jpg

Microscopy of cells expressing fused protein


Bioethanol9.jpg

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