Coding

Part:BBa_K4268007:Design

Designed by: J. Aubrey, J. Alvarenga, L. Buchanan, J. Reyes, D. Yashinski   Group: iGEM22_SUNY_Oneonta   (2022-07-26)


S-TIP37 Tail Fiber-Like Protein


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 NgoMIV site found at 874
    Illegal AgeI site found at 2905
  • 1000
    COMPATIBLE WITH RFC[1000]

Design Notes

The goal of our team's project, Cyanospectre is to create a ghost phage that could be used by synthetic biologists to make genetic engineering of Cyanobacteria easier. We envision that with modification of this virus, it could be used to immobilize target cyanobacteria, or as a viral vector for the delivery of large circuits into a Cyanobacterial chassis.


We selected a cyanophage, S-TIP37 as the model from which the ghost phage could be built. This phage’s natural host is Synechococcus sp WH8109, a marine Cyanobacteria that has been grown in the lab. We selected this phage based on 1) it has a host that can be grown in the lab, 2) the phage has a relatively small genome making capsid proteins easier to identify, and 3) being a T7-like phage, the structure of its neck region is more simple than that of T4-like phages, making the construction of the ghost phage simpler. We envision that with future modeling, the tail fibers of this ghost phage could be modified to make the virus capable of attaching to and delivering capsid contents to a variety of Salt-water Synechococcus sp strains employed in synthetic biology as chassis, such as Synechococcus sp PCC 11901, UTEX 2973, PCC 7942 or 7002.


Although S-TIP 37's genome has been fully sequenced and shared through the National Center for Biotechnology (NCBI) at the National Institutes of Health (NIH) (as NCBI Reference Sequence: NC_048026.1), there is little published research on the virus. Thus resulting in many hypothetical proteins whose functions are unknown and uncertainty regarding the function of proteins that have been provisionally assigned functions. To better characterize these putative genes, bioinformatic tools were used to assess the homology of the S-TIP37 gene with another better characterized T7-like cyanophage, Syn 5. We downloaded the protein sequence of the Tail Fiber-like Protein from S-TIP37 (NCBI Reference Sequence YP_009807536.1), and from Syn 5 (YP_001285455.1). We used NCBI's pBLAST tool to perform pairwise alignments between the putative Tail Fiber-like Protein of S-TIP 37 with that of Syn 5's confirmed one (Pope et al., 2007). Based on feedback we received from an expert in cyanophage genomics, we also elected to perform the same analysis in HMMER.


Figure 1: A BLAST pairwise alignment of putative S-TIP 37 and Syn 5 Tail Fiber-like Protein genes. The protein sequences of the Tail Fiber-like Protein from S-TIP 37 (YP_001285455.1) and from Syn 5 (YP_001285450.1) were downloaded from NCBI and the pBLAST tool was used to perform an alignment.


All alignments express low E-values, indicating that the matches are not random. In addition, we obtained high identity scores for the pairs. Taking into account that the identity scores were mostly greater than 30%, we were able to say with confidence that the structural genes of S-TIP37 we identified were likely to be the correct structural genes needed for the assembly of our ghost phage (Pearson, 2013). We, therefore, concluded that these are homologous proteins and that the selected S-TIP37 gene was likely the Tail Fiber-like Protein for this virus.

We then downloaded the gene sequence for the Tail Fiber-like Protein from the NCBI reference NC_048026.1, gene ID 54998417, and prepared the sequence for synthesis. Due to the large part sequence, IDT was unable to synthesize the entire Tail Fiber-like Protein sequence. The sequence was synthesized as two fragments: Fragment A (1530 nucleotides) and Fragment B (1491 nucleotides). Fragment A would be joined to Fragment B via a KPNA1 restriction site, this must be done before cloning into the level 0 vector as RFP contains two KPNA1 sites.

We codon optimized the sequence for expression in E. coli BL21, as we intend to use E. coli BL21 as the chassis for creating the ghost phage. We also codon-optimized the sequence for cloning using the iGEM RCF10 standard, as well as via Golden Gate using RCF1000. The following illegal sites were removed:

  • BsaI at nucleotide positions 1314-1317
  • BsaI-HFv2 at nucleotide positions 1321-1323
  • BsaI-HFv2 at nucleotide positions 2011-2013
  • BsaI-HFv2 at nucleotide positions 2017-2020


RCF1000 prefix was added to the sequence to facilitate Level 0 cloning via Golden Gate Assembly and the DNA was synthesized by IDT.

Source

The source of this sequence is from the S-TIP37 genome (Gene ID 54998417 from the NCBI reference assembly NC_048026.1) (HOT80_gp40 tail fiber-like protein [Synechococcus T7-like Phage S-TIP37] - Gene - NCBI, n.d.)

References

The S-TIP37 genome was found on GenomeNet

Pearson WR. An introduction to sequence similarity ("homology") searching. Curr Protoc Bioinformatics. 2013 Jun;Chapter 3:Unit3.1. doi: 10.1002/0471250953.bi0301s42. PMID: 23749753; PMCID: PMC3820096.

Pope WH, Weigele PR, Chang J, Pedulla ML, Ford ME, Houtz JM, Jiang W, Chiu W, Hatfull GF, Hendrix RW, King J. Genome sequence, structural proteins, and capsid organization of the cyanophage Syn5: a "horned" bacteriophage of marine synechococcus. J Mol Biol. 2007 May 11;368(4):966-81. doi: 10.1016/j.jmb.2007.02.046. Epub 2007 Feb 22. PMID: 17383677; PMCID: PMC2971696.

U.S. National Library of Medicine. (n.d.). Hot80_gp40 tail fiber-like protein [Synechococcus T7-like phage S-tip37] - gene - NCBI. National Center for Biotechnology Information. From https://www.ncbi.nlm.nih.gov/gene/54998417