Difference between revisions of "Part:BBa K1021005"
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| + | =2019 Fudan-TSI's Characterization= | ||
| + | ==Design== | ||
| + | This year Fudan-TSI uses Cre recombinase as a key component for recombination between reverse-transcribed cDNA and the original gene of interest. After recognizing lox sites, cleavage complex or holiday junction forms and recombination is accomplished. | ||
| + | ==Results== | ||
| + | We have expressed Cre recombinase in ''E.coli'' and analyzed its function by agarose gel. | ||
| + | [[File:Cre+lox Sites.png|none|333px|thumb|'''Fig. 1 The verification of incompatibility between LoxP and different Lox sites.''' | ||
| + | Plasmids containing the Cre-SRC gene were co-transformed respectively with plasmids containing mCherry flanked by a LoxP site and either LoxP, Lox2272 or Lox5171 site. A pair of sequencing primers were used to amplify the mCherry gene. PCR product would be around 1000 bp while fragments cleaved by Cre recombinase would be approximately 250 bp. The positive control group is the PCR product of plasmids containing the original mCherry gene. The negative control group is the PCR product of plasmids containing the Cre-SRC gene only.]] | ||
Latest revision as of 09:54, 13 October 2019
Cre recombinase
The Cre recombinase can be used for site-specific recombination of constructs flanked by lox sites (BBa_J61046). The Cre-Lox recombination system can be used for gene manipulation and as a biosafety mechanism. As a biosafety mechanism, its action is to remove engineered genes flanked by lox sequences.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 356
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 42
- 1000COMPATIBLE WITH RFC[1000]
2019 Fudan-TSI's Characterization
Design
This year Fudan-TSI uses Cre recombinase as a key component for recombination between reverse-transcribed cDNA and the original gene of interest. After recognizing lox sites, cleavage complex or holiday junction forms and recombination is accomplished.
Results
We have expressed Cre recombinase in E.coli and analyzed its function by agarose gel.
Fig. 1 The verification of incompatibility between LoxP and different Lox sites. Plasmids containing the Cre-SRC gene were co-transformed respectively with plasmids containing mCherry flanked by a LoxP site and either LoxP, Lox2272 or Lox5171 site. A pair of sequencing primers were used to amplify the mCherry gene. PCR product would be around 1000 bp while fragments cleaved by Cre recombinase would be approximately 250 bp. The positive control group is the PCR product of plasmids containing the original mCherry gene. The negative control group is the PCR product of plasmids containing the Cre-SRC gene only.