Part:BBa_K3204002
Receptor binding protein of paenibacillus phage HB10c2
This is the putative Receptor binding protein of the paenibacillus phage HB10c2 (family: siphoviridae) with the DNA sequence codon-optimized for the expression in E. coli. It is located in the baseplate phage and binds an unknown receptor on Paenibacillus larvae, the pathogen causing American foulbrood. The binding is very specific to Paenibacillus larvae.
The protein has been identified in silico as the most promising. Modeling of several genes in the cluster of structural proteins of HB10c2 with the webtool Phyre2 has led us to propose the following gene order (gene number in brackets):
(...) - tail length tape measure protein (14) - dit (15) - tal (16) - RBP (17) - (unknown proteins) - lysin (21) - (...)
This order resembles well known clusters of baseplate coding regions for other Siphoviridae phages.
The binding has not been proven with sufficiently reliable experimental data. The experiment suggest that the binding could have worked, but the data is not resilient enough to claim it.
The experiment has been conducted following the protocol of Simpson et al. 2016, where they propose a new assay for the identification of unknown RBPs. We tested E. coli Top10 with and without the plasmid containing the IPTG-inducable putative RBP gene instead of colonies from a gene library. The proteins were plotted onto a nitrocellulose membrane and incubated with P. larvae according to the paper of Simpson et al. 6 of 12 colonies containing the RBP-gene resulted in hardly distinguishable spots of bacteria while none of the 12 colonies without the RBP-gene did. This can bee seen as a hint that the protein could indeed be the the RBP.
The setup and implementation of the experiment need to be further elaborated. Due to time pressure no further experiments have been conducted.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 649
- 1000COMPATIBLE WITH RFC[1000]
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