Part:BBa_K1111007:Design

MMP2 under the control of the pBAD/araC arabinose inducible promoter

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1645
  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  

Design Notes

The construct was obtained by performing a Gibson reaction on the BBa_I746808 part and MMP2-containing plasmid, after an overlap was added by PCR reaction to both of the elements. For the MMP2 CDS, the PCR not only added an overlap complementary to the iGEM part sequence but also a His tag at the beginning of the protein CDS and a 30bp linker at the end of it, so that it would fold properly when expressed followed by the superfolder GFP sequence.

Link for the MMP2 plasmid:http://plasmid.med.harvard.edu/PLASMID/GetCloneDetail.do?cloneid=2849&species=Homo%20sapiens
MMP2 forward primer:5'-ATG CACCACCACCACCACCAC GAG GCG CTA ATG GCC C-3'
MMP2 reverse primer:5'-TTT GAT GCC TGG CTC TAG TA TCA TCA GCA GCC TAG CCA GTC GGA TTT GAT-3'
Backbone forward primer:5'-TCC GAC TGG CTA GGC TGC TGA GAT ACT AGA GCC AGG CAT CAA ATA AAA C-3'
Backbone reverse primer:5'-TAG CGC CTC 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: MMP2 insert

Figure 3: PCR: MMP2 backbone

Gibson Assembly Results:

Figure 4: Gibson Assembly: MMP2-GFP construct

Miniprep Results:

Figure 5: Minipreps:MMP2-GFP constructs

Sequencing alignment results:

Figure 44: MMP2 sequencing overlaps

Source

The MMP2 CDS comes from the species Homo sapiens.

References

MMP2 CDS ordered in a plasmid from plasmid.med.harvard.edu, blasted against NCBI sequence for MMP2. Backbone from Cambridge 2007 part BBa_I746908