Difference between revisions of "Part:BBa K3204002"

Revision as of 09:49, 18 October 2019

Receptor binding protein of paenibacillus phage HB10c2

This is the putative Receptor binding protein of the paenibacillus phage HB10c2 with the DNA sequence codon-optimized for the expression in E. coli. It 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 gene order

(...) - 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 according to the protocol and incubated with P. larvae. Many of the colonies containing the RBP-gene resulted in hardly distingishable spots of bacteria while none of the colonies without the RBP-gene did. The setup and implementation of the experiment need to be further elaborated. Due to time pressure no further experiments were conducted.

Sequence and Features