Composite

Part:BBa_K4268018:Design

Designed by: J. Aubrey, J. Alvarenga, L. Buchanan, J. Reyes, D. Yashinski     Group: iGEM22_SUNY_Oneonta   (2022-09-30)


Biotin Tagged Capsid and Core Multi-Transcriptional Unit


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 3806
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 3806
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 5170
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 3806
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 3806
    Illegal NgoMIV site found at 3446
    Illegal NgoMIV site found at 3585
    Illegal NgoMIV site found at 4541
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The Biotin tagged Capsid and Core Multi-Transcriptional Unit (MTU) codes for the putative proteins that make up the icosahedral head (Internal Core, Biotin Tagged Capsid, and Capsid Assembly Protein) of the head component of the S-TIP 37 capsid. The component transcriptional units and basic parts were designed to be compatible with Type IIS assembly via Golden gate cloning. The MTU is a level 2 assembly of multiple level 1 transcriptional units' and permits modular assembly so the user may customize the part as needed. The component transcriptional unit parts have been uploaded to the parts repository under the parts numbers BBa_K4268014, BBa_K4268011, BBa_K4268009.


A dummy part (Type IIS pSB1K04-DY) designed by Team UofUppsala 2020 is also included. The purpose of this part is to provide the 5’ and 3’ flanking sites necessary for assembly. This part can be substituted with another coding part based on the needs of the user.

Figure 1: SBOL representation of the Biotin tagged Capsid and Core MTU.


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 1 vector. Therefore, for our level 2 MTU design, it will be critical to clone the level 1 parts into the appropriate level 1 vector. To obtain this design, the level 1 parts should be assembled into the iGEM standard golden gate vectors as in table 1.

Figure 1: Cloning of level 1 transcriptional units into level 1 golden gate vectors to create BBa_K4268018.

For the same reasons, when assembling the level 2 component, care should be taken to select vectors that will yield the desired component order.

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.