Composite

Part:BBa_K523025:Design

Designed by: Chris French, Eugene Fletcher, Sylvia Ispasanie   Group: iGEM11_Edinburgh   (2011-10-28)
Revision as of 12:19, 28 October 2011 by Allancrossman (Talk | contribs) (→‎Design Notes)

Plac + RBS + cxnA (cex + cenA fusion)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1148
    Illegal NotI site found at 2846
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2759
    Illegal XhoI site found at 2257
    Illegal XhoI site found at 2506
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 781
    Illegal NgoMIV site found at 1154
    Illegal NgoMIV site found at 1656
    Illegal NgoMIV site found at 2037
    Illegal NgoMIV site found at 2962
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1201
    Illegal SapI.rc site found at 1284


Design Notes

cex normally contains domains GH10 (exoglucanase module) plus CBM2 (cellulose binding module). cenA contains a homologous CBM2 domain plus GH6 (endoglucanase module). It was therefore decided to fuse the two proteins at the CBM to make the new protein, designated cxnA, as follows:

  • GH10 + CBM2 + GH6

By analysis of the matching CBM2 domains, it was determined that an NcoI site could easily be introduced into both at the same place without changing any amino acids.

BBa_K523016 (Plac + lacZ&alpha + RBS + cex) was amplified using forward primer pSBNX3insf2 (vector-based) and a new reverse primer which introduces an NcoI site in the CBM2 domain of cex.

cenA was amplified using a new forward primer which likewise introduces an NcoI site in the corresponding location of the CBM2 domain, and reverse primer pSBNX3insr2 (vector-based).

primer cenAfNcoI: c ctg cca tgg aac ggc agc atc ccg acc
primer cexrNcoI: c gtt cca tgg ggc gtt gcg gac cgt cac g

The two PCR products were purified, digested with NcoI and ligated, and the ligation was used as template for PCR with primers pSB1NX2insf2 and pSB1NX3insr2. Two major products were obtained. The larger, around 3.1 kb, was presumed to be correct, and was purified from the gel. The smaller, around 2.1 kb, was presumed to be a mixture of Plac-lacZ'-cenA and Plac-lacZ'-cex from intact template which had made it through the purification.

The purified insert was digested with EcoRI/PstI and ligated into pSB1C3. White colonies were tested for Cex activity on MUC plates. Positive clones were miniprepped and tested for insert size. Clones were further analysed by digestion with NcoI/PstI to check that the NcoI site was present. They were then tested for CenA activity on CMC plates.

It is therefore presumed, subject to confirmation of sequence, that clones which passed these tests represent the fusion of cex and cenA (i.e. Plac + lacZα + RBS + cxnA) and that this has both enzyme activities.

Source

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References