Composite

Part:BBa_K1111009:Design

Designed by: Luisa Spisak, Marcelle Isaline Arrigo   Group: iGEM13_EPF_Lausanne   (2013-09-19)
Revision as of 14:51, 4 October 2013 by Miarrigo (Talk | contribs) (→‎Design Notes)

Gelatinase under pBAD/araC arabinose inducible promoter


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 3101
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
    Illegal PstI site found at 3101
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2634
    Illegal BglII site found at 2685
    Illegal BamHI site found at 1144
    Illegal BamHI site found at 3028
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 3101
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 3101
    Illegal NgoMIV site found at 2330
    Illegal AgeI site found at 979
    Illegal AgeI site found at 2703
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1517
    Illegal BsaI.rc site found at 2234
    Illegal SapI site found at 961
    Illegal SapI.rc site found at 3169


Design Notes

The gelE CDS was first isolated from the E. faecalis genome by PCR and the primers used for that also added a His tag at the beginning of the CDS and a linker at the end. Then, a Gibson assembly was performed with our amplified part BBa_I746908 to insert gelE CDS in between the pBAD/araC promoter and superfolder GFP. Bacteria were then transformed with the plasmid, who was isolated and sent for sequencing. This last process allowed us to see that a STOP codon had appeared just before the GFP sequence, after the linker. However, the protein should still be isolate-able since a His-tag was added to it.

Link for the NCBI GelE CDS:http://www.ncbi.nlm.nih.gov/nuccore/D85393.1
MMP2 forward primer:5'--3'
MMP2 reverse primer:5'--3'
Backbone forward primer:5'--3'
Backbone reverse primer:5'--3'

Figure 1: Gibson assembly: Protein with GFP

















PCR Results:

Figure 2: PCR: GelE insert
Figure 3: PCR: GelE backbone



























Gibson Assembly Results:

Figure 4: Gibson Assembly: GelE-GFP construct























Miniprep Results:

Figure 5: Minipreps:GelE-GFP constructs





















Sequencing alignment results:

Figure 6: GelE sequencing overlaps











Source

The gelE CDS was extracted from Gram positive bacterium E.faecalis' genomic DNA.

References

GelE CDS extracted by PCR from the genomic DNA of E. faecalis and blasted against the GelE NCBI sequence. Backbone from Camridge 2007 part BBa_I746908.