Difference between revisions of "Part:BBa K1607011:Design"
(→Source) |
(→References) |
||
| (2 intermediate revisions by the same user not shown) | |||
| Line 12: | Line 12: | ||
The eGFP CDS was obtained using PCR. | The eGFP CDS was obtained using PCR. | ||
Primers: | Primers: | ||
| + | |||
ggaggcggaggcagcggtggcggtggaagcATGGTGAGCAAGGGCga (eGFP-F) | ggaggcggaggcagcggtggcggtggaagcATGGTGAGCAAGGGCga (eGFP-F) | ||
| Line 17: | Line 18: | ||
The eGFP CDS and the scFv CDS were connected by SOE PCR | The eGFP CDS and the scFv CDS were connected by SOE PCR | ||
| + | |||
Primers: | Primers: | ||
| + | |||
step1: | step1: | ||
| + | |||
CGGAATTCatgGGTTCTACTCAAG (SCFV-F) | CGGAATTCatgGGTTCTACTCAAG (SCFV-F) | ||
| Line 32: | Line 36: | ||
Standard Biobrick prefix and suffix were then added to the sequence by PCR, replacing the EcoRI site and XbaI site which were originally designed for yeast expression. | Standard Biobrick prefix and suffix were then added to the sequence by PCR, replacing the EcoRI site and XbaI site which were originally designed for yeast expression. | ||
| + | |||
Primers: | Primers: | ||
| + | |||
GAATTCGCGGCCGCTTCTAGatgGGTTCTACTCAAGttt (F) | GAATTCGCGGCCGCTTCTAGatgGGTTCTACTCAAGttt (F) | ||
| Line 38: | Line 44: | ||
===Design Notes=== | ===Design Notes=== | ||
| + | This part is designed for FRET analysis. An Mcherry will be fused with the antigen, and by measuring the FRET efficiency we can detect the antibody-antigen interaction. | ||
===References=== | ===References=== | ||
| + | [1] Zhou H, Zha Z, Liu Y, et al. Structural Insights into the Down-regulation of Overexpressed p185her2/neu Protein of Transformed Cells by the Antibody chA21[J]. Journal of Biological Chemistry, 2011, 286(36): 31676-31683. | ||
Latest revision as of 13:45, 17 September 2015
The SCFV of anti-p185her2/neu antibody chA21 linked with EGFP by a (Gly4Ser)3 linker
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 370
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Source
The scFv CDS was synthesized by LCR assmebly using 36 oilgos
The eGFP CDS was obtained using PCR. Primers:
ggaggcggaggcagcggtggcggtggaagcATGGTGAGCAAGGGCga (eGFP-F)
GGTCTAGAGGCTTGTACAGCTCGTCCAT (eGFP-R)
The eGFP CDS and the scFv CDS were connected by SOE PCR
Primers:
step1:
CGGAATTCatgGGTTCTACTCAAG (SCFV-F)
gctgcctccgcctccagaaccgccaccgccAGATTGACAATCTTCagaaga (SCFV-R)
ggaggcggaggcagcggtggcggtggaagcATGGTGAGCAAGGGCga (eGFP-F)
GGTCTAGAGGCTTGTACAGCTCGTCCAT (eGFP-R)
step2:
SCFV-F and eGFP-R
Standard Biobrick prefix and suffix were then added to the sequence by PCR, replacing the EcoRI site and XbaI site which were originally designed for yeast expression.
Primers:
GAATTCGCGGCCGCTTCTAGatgGGTTCTACTCAAGttt (F)
TACTAGTAGCGGCCGCTGCAGCTTGTACAGCTCGTCCAT(R)
Design Notes
This part is designed for FRET analysis. An Mcherry will be fused with the antigen, and by measuring the FRET efficiency we can detect the antibody-antigen interaction.
References
[1] Zhou H, Zha Z, Liu Y, et al. Structural Insights into the Down-regulation of Overexpressed p185her2/neu Protein of Transformed Cells by the Antibody chA21[J]. Journal of Biological Chemistry, 2011, 286(36): 31676-31683.