Part:BBa_K4268020:Design
S-TIP 37 Ghost Phagemid with Biotin-tagged Capsid
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 3810
Illegal PstI site found at 7523 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 6161
Illegal NheI site found at 10697
Illegal PstI site found at 3810
Illegal PstI site found at 7523 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 5174
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 3810
Illegal PstI site found at 7523 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 3810
Illegal PstI site found at 7523
Illegal NgoMIV site found at 3450
Illegal NgoMIV site found at 3589
Illegal NgoMIV site found at 4545
Illegal NgoMIV site found at 12194
Illegal AgeI site found at 14225 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
The S-TIP 37 Ghost Phagemid with Biotin-tagged Capsid codes for the putative proteins that make up the icosahedral head (Internal ore, Biotin-tagged Capsid, and Capsid Assembly Protein), neck region (Tail Tubular A, B, and the Head-Tail Connector Protein), and tail fibers (Tail Fiber-like Protein) of S-TIP 37. The component multi-transcriptional units (MTU), transcriptional units (TU), and basic parts were designed to be compatible with Type IIS assembly via Golden Gate cloning. The phagemid is a level 3 assembly of multiple level 2 MTUs' and permits modular assembly so the user may customize the part as needed. These parts have been uploaded to the parts repository under the parts numbers BBa_K4268016 and BBa_K4268018.
Two dummy parts (Type IIS pSB3C13-DY, and Type IIS pSB3C14-DY) designed by Team UofUppsala 2020 are also included. The purpose of these parts is to provide the 5’ and 3’ flanking sites necessary for assembly. These parts can be substituted with another coding part based on the needs of the user.
One of the main concerns in designing the level 2 MTUs is gene order. Studies have demonstrated that the order that genes are placed within an operon can impact the rate of translation, and therefore, the level of expression (Wells et. al. 2016, Lim et. al. 2011). While each gene in the MTU will have its own promoter and is therefore not organized within an operon, gene order may impact expression, and will likely require optimization. For the initial iteration of the MTU design, we organized the genes in the MTU so that genes requiring the highest levels of expression were not located on the “ends” of the MTU.
For assembly, the order in which TUs will be assembled into MTUs will be dependent on the 5' and 3' flanking sites present on each TU. These sites are provided by the level 2 vector. Therefore, for our level 3 phagemid design it will be critical to clone the level 2 parts into the appropriate level 2 vectors. Team UofUppsala 2020 identified an illegal BsaI restriction site in the pSB3C0# pEven Loop vectors that are the standard vectors for level 3 cloning. The team redesigned these vectors to create the pSB3C1# series, using site-directed mutagenesis to introduce a G to C point mutation at position 290 to remove this site. Therefore, for assembly of this construct, we recommend using the pSB3C1# series vectors, or if not possible, utilize site-directed mutagenesis as detailed on the parts pages for this vector series to remove the illegal site from the original set. To obtain the design of this phagemid, the level 2 parts should be assembled into the iGEM standard Golden Gate vectors as in table 1.
Source
The source of the coding sequences included in this part are from the S-TIP37 genome (from the NCBI reference assembly NC_048026.1).
The remaining components are from the parts repository and originate in E. coli or T7 bacteriophage, or were designed in silico.
References
Lim HN, Lee Y, Hussein R. Fundamental relationship between operon organization and gene expression. Proc Natl Acad Sci U S A. 2011 Jun 28;108(26):10626-31. doi: 10.1073/pnas.1105692108. Epub 2011 Jun 13. PMID: 21670266; PMCID: PMC3127940.
Wells JN, Bergendahl LT, Marsh JA. Operon Gene Order Is Optimized for Ordered Protein Complex Assembly. Cell Rep. 2016 Feb 2;14(4):679-685. doi: 10.1016/j.celrep.2015.12.085. Epub 2016 Jan 21. PMID: 26804901; PMCID: PMC4742563.