Difference between revisions of "Part:BBa K4268003:Design"

Latest revision as of 16:02, 7 October 2022

S-TIP37 Capsid Protein with Biotin Tag

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.

Whilst developing our project, we came upon a need within the iGEM Community for immobilizing cyanobacteria and thus we modified our phagemid to align with this need. Biotin-streptavidin interactions result in strong attraction between two. By adding a biotin tag to the Capsid Protein (gp 33), it is projected that the capsid head will develop with the tags, creating a biotin-lined capsid head. The phagemid will attach itself to cyanobacteria and in the presence of a streptavidin-coated plate, the phagemid will bind itself to it whilst still maintaining its attachment to the cyanobacteria (Figure 1).

Figure 1: A visual depiction of the biotin-strepavidin interaction when applied to our phagemid.

The Capsid Protein with Biotin Tag was created using bioinformatic tools. The gene sequence for the Capsid Protein was downloaded from the NCBI reference NC_048026.1, gene ID 54998410, and its stop codon was removed. The Biotin Tag had its illegal sites removed before its addition to the end of the Capsid Protein. It was ensured that the sequence was codon optimized 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:

• NotI at nucleotide positions 1-3 of the biotin tag only
• Capsid Protein had no illegal sites

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 54998410 from the NCBI reference assembly NC_048026.1) (HOT80_gp33 Capsid 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_gp33capsid protein [Synechococcus T7-like phage S-tip37] - gene - NCBI. National Center for Biotechnology Information. From https://www.ncbi.nlm.nih.gov/gene/54998410