Difference between revisions of "Part:BBa K1111007:Design"

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Link for the MMP2 plasmid:http://plasmid.med.harvard.edu/PLASMID/GetCloneDetail.do?cloneid=2849&species=Homo%20sapiens <BR>
 
Link for the MMP2 plasmid:http://plasmid.med.harvard.edu/PLASMID/GetCloneDetail.do?cloneid=2849&species=Homo%20sapiens <BR>
 
MMP2 forward primer:5'-ATG CACCACCACCACCACCAC GAG GCG CTA ATG GCC C-3' <BR>
 
MMP2 forward primer:5'-ATG CACCACCACCACCACCAC GAG GCG CTA ATG GCC C-3' <BR>
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MMP2 reverse primer:5'-TTT GAT GCC TGG CTC TAG TA TCA TCA GCA GCC TAG CCA GTC GGA TTT GAT-3' <BR>
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MMP2 reverse primer:5'-ACC ACC AGA TTG AAA ATA CAA ATT TTC ACC GCA GCC TAG CCA GTC GGA TTT GAT-3' <BR>
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Backbone forward primer:5'-TCC GAC TGG CTA GGC TGC TGA GAT ACT AGA GCC AGG CAT CAA ATA AAA C-3' <BR>
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Backbone forward primer:5'-GGT GAA AAT TTG TAT TTT CAA TCT GGT GGT GAT GCG TAA AGG CGA AGA GCT-3' <BR>
 
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' <BR>
 
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' <BR>
 
[[Image: GFP construct.PNG|thumb|200px|left|Figure 1: Gibson assembly: Protein with GFP]]
 
[[Image: GFP construct.PNG|thumb|200px|left|Figure 1: Gibson assembly: Protein with GFP]]
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[[Image: MMP2 insert PCR.JPEG|thumb|200px|left|Figure 2: PCR: MMP2 insert]]
 
[[Image: MMP2 insert PCR.JPEG|thumb|200px|left|Figure 2: PCR: MMP2 insert]]
 
[[Image: MMP2 backbone PCR.JPEG|thumb|200px|left|Figure 3: PCR: MMP2 backbone]]
 
[[Image: MMP2 backbone PCR.JPEG|thumb|200px|left|Figure 3: PCR: MMP2 backbone]]
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Gibson Assembly Results: <BR>
 
Gibson Assembly Results: <BR>
 
[[Image: MMP2 Gibson.JPEG|thumb|200px|left|Figure 4: Gibson Assembly: MMP2-GFP construct]]
 
[[Image: MMP2 Gibson.JPEG|thumb|200px|left|Figure 4: Gibson Assembly: MMP2-GFP construct]]
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Miniprep Results: <BR>
 
Miniprep Results: <BR>
 
[[Image: MMP2 minipreps.JPEG|thumb|200px|left|Figure 5: Minipreps:MMP2-GFP constructs]]
 
[[Image: MMP2 minipreps.JPEG|thumb|200px|left|Figure 5: Minipreps:MMP2-GFP constructs]]
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Sequencing alignment results: <BR>
 
Sequencing alignment results: <BR>
 
[[Image: MMP2 sequence overlap.JPEG|thumb|200px|left|Figure 44: MMP2 sequencing overlaps]]
 
[[Image: MMP2 sequence overlap.JPEG|thumb|200px|left|Figure 44: MMP2 sequencing overlaps]]

Latest revision as of 14:51, 4 October 2013

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'-ACC ACC AGA TTG AAA ATA CAA ATT TTC ACC GCA GCC TAG CCA GTC GGA TTT GAT-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'-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