Part:BBa_K781006

[R0010][B0034] - RFP-FliC Deletion Chimera

This is the modified RFP protein obtained from J04450, that has been incorporated into the E. coli flagellin as a deletion.

This part can be used as a template for making other PCR overlap insertions. After transforming the product, and digesting the template plasmid with Dpn1, any template that has not been successfully digested will form red colonies.

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.

Fig. 1: 3D model of an RFP protein (Red) inserted into the sequence of the flagellin from Salmonella Typhymurium (Green) in the place of the D3 variable domain. PDB IDs: 1GGX, 1UCU

Fig. 2: TOP10 E. coli expressing J04450, the RFP insertion chimeric flagellin (K781001) and the RFP deletion chimeric flagellin (K781006).

Fig. 3: The fluorescence spectra of TOP10 E. coli cells expressing the RFP flagellin insertion K781001 and deletion K781006 constructs compared against J04450, the corresponding RFP control.

Fig. 4 The motility assay for the K781006 construct compared against controls expressing no flagellin construct and a non-chimeric flagellin construct. Inoculations made on 0.25% swimming agar.

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.

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

References:
1. [http://www.biotechniques.com/BiotechniquesJournal/2010/June/Overlap-extension-PCR-cloning-a-simple-and-reliable-way-to-create-recombinant-plasmids/biotechniques-280116.html LINK] 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 Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1884
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 529
  Illegal AgeI site found at 1362
  Illegal AgeI site found at 1474
  
• 1000
  COMPATIBLE WITH RFC[1000]