Part:BBa_K1111009:Design

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
GelE forward primer:5'-ATG CACCACCACCACCACCAC AAG GGA AAT AAA ATT TTA TAC CAT TTT A-3'
GelE reverse primer:5'-ACC ACC AGA TTG AAA ATA CAA ATT TTC ACC TTC ATT GAC CAG AAC AGA TTC-3'
Backbone forward primer:5'-GGT GAA AAT TTG TAT TTT CAA TCT GGT GGT GAT GCG TAA AGG CGA AGA GCT-3'
Backbone reverse primer:5'-ATT TCC CTT GTG GTG GTG GTG GTG GTG CAT TAG TAT TTC TCC TCT TTC TCT AGT AGC TAG-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.