Difference between revisions of "Part:BBa K781001"

Revision as of 14:24, 4 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 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.

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

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

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.

References: 1. Anton V. Bryksin and Ichiro Matsumura. (2010) Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. BioTechniques, Vol. 48, No. 6, pp. 463–465 </html>

Sequence and Features