Coding

Part:BBa_K1201000:Design

Designed by: krunal polra   Group: iGEM13_Westminster   (2013-09-23)

Derived from serratia marcescens


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 NgoMIV site found at 1227
    Illegal NgoMIV site found at 1678
    Illegal AgeI site found at 274
    Illegal AgeI site found at 1381
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

ChiA was received in an IPTG inducible expression plasmid, pQE-70. The parts contain a pstI site. This site was removed via a site directed mutagenesis using overlapping primers. In the gel below lane 3 shows one half of ChiA which was amplifies using a forward primer and a reverse muatgenic primer. Lane 4 shows the second half of ChiA which was amplifies using a reverse primer and a forward mutagenic primer.

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In the gel below in lane 2 is the full gene (ChiA). The two halves were mixed together and allow to self primer giving a template. This template was then amplified using the outer primers. ChiA was then gel extracted and ligated to pSB1C3.

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Characterization

We used the Chitin Azure Assay to test the activity of the chitinase genes.

In the diagram on the left is the whole cell culture of E. Coli Top10 strain expressing IPTG induced ChiA in pQE-70, ChiB in pQE-60 and ChiC in pQE-60. 200uL of each culture was added to 600uL of chitin azure assay substrate. The reaction was incubated at 37C for 72 hours.

400ul of ChiA and 400uL of chinin azure assay substrate were also incubated at 37C for 72 hours. ChiA did not produce a reaction when tested on its own, but a positive result was received when all three Chi genes were in one reaction mixture.

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Source

Source organism: Serratia marcescens. The gene in a plasmid was gifted by Dr. Frank Sargent from The University of Dundee

References

Vaaje-Kolstad, G., Horn, S.J., Sørlie, M., Eijsink, V.G.H., (2013). The chitinolytic machinery

of Serratia marcescens--a model system for enzymatic degradation of recalcitrant

polysaccharides. The FEBS Journal. 280 (13), 3028. http://onlinelibrary.wiley.com/doi/

10.1111/febs.12181/pdf


http://www.sanger.ac.uk/resources/downloads/bacteria/serratia-marcescens.html