Difference between revisions of "Part:BBa K3204002"
 
(6 intermediate revisions by the same user not shown)
Line 3: Line 3:
 
<partinfo>BBa_K3204002 short</partinfo>
 
<partinfo>BBa_K3204002 short</partinfo>
  
−
This is the putative Receptor binding protein of the paenibacillus phage HB10c2 (family: siphoviridae) with the DNA sequence codon-optimized for the expression in <i>E. coli</i>. It is located in the baseplate phage and binds an unknown receptor on <i>Paenibacillus larvae</i>, the pathogen causing American foulbrood. The binding is very specific to <i>Paenibacillus larvae</i>.
+
This is the putative receptor binding protein (RBP) of the Paenibacillus phage HB10c2 (family: <i>Siphoviridae</i>, NCBI accession number: NC_028758) with the DNA sequence codon-optimized for the expression in <i>E. coli</i> with the tool from Thermo Scientific. It is located as a homotrimer in the baseplate of the phage and highly specifically binds a yet unknown receptor on the surface of <i>Paenibacillus larvae</i>, the pathogen causing American foulbrood. The binding is very specific to <i>Paenibacillus larvae</i>.
  
−
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):
+
===Characterization===
  
−
(...) - tail length tape measure protein (14) - dit (15) - tal (16) - RBP (17) - (unknown proteins) - lysin (21) - (...)
+
The protein has been identified in silico as the most promising among candidates. Modeling of several genes encoded in the cluster of structural proteins in the genome of HB10c2 with the webtool Phyre2 has helped us identify those proteins with some certainty. Phyre 2 has predicted similarities to already characterized proteins in the baseplate of other phages, whose respective functions remain the same. The order of the genes encoded in the baseplate region of the genome resembles the order of the baseplate genes of other phages, with some slight changes. The baseplate encoding region always follows the tmp and is ended with a lysis. It always contains dit, tal and RBP[1]. Following you can see the baseplate gene order of HB10c2 (gene number in brackets):
  
−
This order resembles well known clusters of baseplate coding regions for other Siphoviridae phages.
+
(...) - tmp(14) - dit(15) - tal(16) - RBP(17) - (unknown proteins) - lysin(21) - (...)
  
−
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.
+
tmp - tail length tape measure protein; dit - distal tail protein; tal - tail-associated lysin protein; RBP - receptor binding protein
  
−
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 <i>E. coli</i> 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 <i>P. larvae</i> 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.
+
Further modeling with Rosetta3 has resulted in a trimeric and thus far better prediction, which accurately resembles the distal structure of know RBPs with the binding loop region, while the anchoring region still could not be modeled satisfyingly, probably due to a lack of structural data for these type of proteins.
  
−
The setup and implementation of the experiment need to be further elaborated. Due to time pressure no further experiments have been conducted.  
+
The binding of the phage HB10c2 to <i>P. larvae</i> has been confirmed via plaque assay. The existence of the gene in the genome has been proven by PCR, which resulted in DNA-fragments of the exact size of the RBP-gene.
 +
 
 +
A wetlab experiment has been conducted following the protocol of Simpson et al. 2016 [2], where they propose a new assay for the identification of unknown RBPs. We tested <i>E. coli</i> Top10 with and without the IPTG-inducible plasmid containing the putative RBP gene (instead of colonies from a gene library, as they propose it). The proteins were blotted onto a nitrocellulose membrane and incubated with <i>P. larvae</i>. 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.
 +
 
 +
Although the experimental data alone is not resilient enough to prove it, it suggests that the candidate protein has P. larvae binding activity. Combined with the modeling results we conclude that this sequence indeed encodes the RBP of HB10c2 .
  
−
<!-- Add more about the biology of this part here
 
−
===Usage and Biology===
 
  
 
<!-- -->
 
<!-- -->
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3204002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3204002 SequenceAndFeatures</partinfo>
 +
<br>
 +
==References==
 +
[1] A. S. A. Dowah and M. R. J. Clokie, ‘Review of the nature, diversity and structure of bacteriophage receptor binding proteins that target Gram-positive bacteria’, Biophys. Rev., vol. 10, no. 2, pp. 535–542, Apr. 2018.
  
 +
[2] D. J. Simpson, J. C. Sacher, and C. M. Szymanski, ‘Development of an Assay for the Identification of Receptor Binding Proteins from Bacteriophages’, Viruses, vol. 8, no. 1, Jan. 2016.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 21:59, 21 October 2019


Receptor binding protein of paenibacillus phage HB10c2

This is the putative receptor binding protein (RBP) of the Paenibacillus phage HB10c2 (family: Siphoviridae, NCBI accession number: NC_028758) with the DNA sequence codon-optimized for the expression in E. coli with the tool from Thermo Scientific. It is located as a homotrimer in the baseplate of the phage and highly specifically binds a yet unknown receptor on the surface of Paenibacillus larvae, the pathogen causing American foulbrood. The binding is very specific to Paenibacillus larvae.

Characterization

The protein has been identified in silico as the most promising among candidates. Modeling of several genes encoded in the cluster of structural proteins in the genome of HB10c2 with the webtool Phyre2 has helped us identify those proteins with some certainty. Phyre 2 has predicted similarities to already characterized proteins in the baseplate of other phages, whose respective functions remain the same. The order of the genes encoded in the baseplate region of the genome resembles the order of the baseplate genes of other phages, with some slight changes. The baseplate encoding region always follows the tmp and is ended with a lysis. It always contains dit, tal and RBP[1]. Following you can see the baseplate gene order of HB10c2 (gene number in brackets): 

(...) - tmp(14) - dit(15) - tal(16) - RBP(17) - (unknown proteins) - lysin(21) - (...)

tmp - tail length tape measure protein; dit - distal tail protein; tal - tail-associated lysin protein; RBP - receptor binding protein

Further modeling with Rosetta3 has resulted in a trimeric and thus far better prediction, which accurately resembles the distal structure of know RBPs with the binding loop region, while the anchoring region still could not be modeled satisfyingly, probably due to a lack of structural data for these type of proteins.

The binding of the phage HB10c2 to P. larvae has been confirmed via plaque assay. The existence of the gene in the genome has been proven by PCR, which resulted in DNA-fragments of the exact size of the RBP-gene.

A wetlab experiment has been conducted following the protocol of Simpson et al. 2016 [2], where they propose a new assay for the identification of unknown RBPs. We tested E. coli Top10 with and without the IPTG-inducible plasmid containing the putative RBP gene (instead of colonies from a gene library, as they propose it). The proteins were blotted onto a nitrocellulose membrane and incubated with P. larvae. 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.

Although the experimental data alone is not resilient enough to prove it, it suggests that the candidate protein has P. larvae binding activity. Combined with the modeling results we conclude that this sequence indeed encodes the RBP of HB10c2 .


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 649
  • 1000
    COMPATIBLE WITH RFC[1000]


References

[1] A. S. A. Dowah and M. R. J. Clokie, ‘Review of the nature, diversity and structure of bacteriophage receptor binding proteins that target Gram-positive bacteria’, Biophys. Rev., vol. 10, no. 2, pp. 535–542, Apr. 2018.

[2] D. J. Simpson, J. C. Sacher, and C. M. Szymanski, ‘Development of an Assay for the Identification of Receptor Binding Proteins from Bacteriophages’, Viruses, vol. 8, no. 1, Jan. 2016.