Difference between revisions of "Part:BBa K781001"
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This is the modified RFP protein obtained from J04450, that has been incorporated into the E. coli flagellin as an insertion. | This is the modified RFP protein obtained from J04450, that has been incorporated into the E. coli flagellin as an insertion. | ||
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<html> | <html> | ||
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<img src="https://static.igem.org/mediawiki/2012/9/9c/Rfp_flic_results_1.JPG" align="right" width=500px> | <img src="https://static.igem.org/mediawiki/2012/9/9c/Rfp_flic_results_1.JPG" align="right" width=500px> | ||
<img src="https://static.igem.org/mediawiki/2012/b/b3/Rfp_spectra.png" width=900px> | <img src="https://static.igem.org/mediawiki/2012/b/b3/Rfp_spectra.png" width=900px> | ||
| + | <img src="https://static.igem.org/mediawiki/parts/thumb/e/e7/D3_RFP.JPG/800px-D3_RFP.JPG" align="right" width=500px> | ||
<br> | <br> | ||
References:<br> | References:<br> | ||
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<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
| + | This part can be used as a template for making other PCR overlap insertions [1]. After transforming the product, and digesting the template plasmid with Dpn1, any template that has not been successfully digested will form red colonies. | ||
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| + | The overlap extension can be done using the following standard: | ||
| + | Prefix Overlapping region Linker | ||
| + | GAATTC GCGGCCGC ACTAGT GATAACGATGGGAAGTATTACGCAGTAACA ACCACAGGAGGTGGAGGTTC ... | ||
| + | Linker Overlapping region Suffix | ||
| + | ...--- Insert --- GGTGGATCAGGTGGAACTTCA ACAGTGACAATGGCGACTGGAGCAACG GCTAGC GCGGCCG CTGCAG | ||
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| + | After Digest: | ||
| + | Prefix Overlapping region Linker | ||
| + | CTAGT GATAACGATGGGAAGTATTACGCAGTAACA ACCACAGGAGGTGGAGGTTC ... | ||
| + | 3'-A CTATTGCTACCCTTCATAATGCGTCATTGT TGGTGTCCTCCACCTCCAAG ... | ||
| + | Linker Overlapping region Suffix | ||
| + | ...--- Insert --- GGTGGATCAGGTGGAACTTCA ACAGTGACAATGGCGACTGGAGCAACG G-3' | ||
| + | ...--- Insert --- CCACCTAGTCCACCTTGAAGT TGTCACTGTTACCGCTGACCTCGTTGC CCTAG | ||
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Revision as of 13:49, 7 October 2012
[R0010][B0034] - RFP-FliC Insertion Chimera
This is the modified RFP protein obtained from J04450, that has been incorporated into the E. coli flagellin as an insertion.
This part was successfully characterized and determined to show expression of the red fluorescent protein from J04500. As can be seen below, there appears to be less detectable fluorescence in cell cultures expressing the different types of chimeras. This result can be expected for a number of possible reasons.
- The chimeric protein may not be as stable as the free RFP.
- The RFP may be polymerizing in flagella, resulting in a quenching effect as the fluorescent light may be absorbed by other RFP molecules arranged in close proximity.
- The flagellin domains may hinder proper folding of the RFP. This may be why the more constrained deletion variant is showing less expression compared to the insertion variant, which has more flexibility.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2173
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 530
Illegal AgeI site found at 938
Illegal AgeI site found at 1552
Illegal AgeI site found at 1664 - 1000COMPATIBLE WITH RFC[1000]