To create the phagemid, we began by designing the basic parts needed to create the S-TIP37 capsid. We obtained the gene sequences for the capsid protein, head-tail connector, capsid assembly protein, tail tubular protein A and B, internal core protein, and the tail fiber-like protein from S-TIP37 from NCBI, as described on our Project Design page. We also designed a version of the capsid protein modified by the addition of a linker and a biotin tag for a partnership with Team MSP-Maastricht (Figure 1). More details on the design of the tagged capsid protein can be found on our Partnership page.
Figure 1. An alternate version of the cyanophage capsid includes a biotin tag
These sequences were optimized for expression in E. coli BL21 using the IDT DNA Codon Optimization tool, and then checked for compatibility with the iGEM RCF1000 assembly standard using NEBCutter 3.0. Silent mutations were introduced to remove any illegal sites and flanking sequences were added to the 5’ and 3’ ends to facilitate Golden Gate cloning. All domesticated sequences were then synthesized by IDT or Twist Bioscience. Once the synthesized DNAs were received, we used Golden Gate assembly to clone the parts into the level zero vector pSB1C00.
Samples of the ligation reactions were used to transform E. coli DH5α cells. After selection on LB plates containing chloramphenicol, potential positive colonies were screened using colony PCR. The expected molecular weight of the PCR product is 305 bp plus the size of the coding sequence. Based on the size of the PCR products visualized by agarose gel electrophoresis, we successfully verified cloning of six of these basic parts into the pSB1C00 vector by colony PCR (Figure 2).
Figure 2: Colony PCR confirming Level 0 cloning of six S-TIP37 capsid proteins. The PCR products observed for many colonies matched the expected size. Capsid = 1026 bp, Capsid with biotin tag = 1086 bp, Head-tail connector = 1878 bp, Tail tubular A = 921 bp, Tail tubular B = 3057 bp, Capsid Assembly = 1073 bp, RFP reporter device = 1374 bp.
We made several attempts to clone the tail fiber-like and the internal core proteins, but so far have been unsuccessful. We believe that we are having difficulty cloning these parts due to their size. The tail fiber-like protein gene is 3022 bp, and the internal core protein gene is 3619 bp. Both parts are too large to be synthesized as a single piece of DNA. Our strategy for overcoming this was to have these parts created as two pieces of DNA, and then use an internal unique restriction site to join the fragments together. We are continuing cloning attempts and have begun exploring alternative approaches to obtaining these large parts.
Simultaneously, we began to work on cloning the Level 1 transcriptional units needed for assembly of the phagemid. We elected to use a T7-inducible promoter system to regulate the expression of the capsid proteins in E. coli BL21. We selected a T7 promoter library from the iGEM parts repository which includes promoters with varying levels of strength (Table 1). We elected to use a common RBS (BBa_B0034) and a common terminator sequence (BBa_B0015) for each transcriptional unit (Figure 3).
| Part Number | Strength compared to consensus |
Parts Sequence |
|---|---|---|
| BBa_R0085 | 1 | TAA TAC GAC TCA CTA TAG GGA GA |
| BBa_R0180 | 0.72 | TTA TAC GAC TCA CTA TAG GGA GA |
| BBa_R0181 | 0.5 | GAA TAC GAC TCA CTA TAG GGA GA |
| BBa_R0182 | 0.3 | TAA TAC GTC TCA CTA TAG GGA GA |
| BBa_R0183 | 0.09 | TCA TAC GAC TCA CTA TAG GGA GA |
We ordered the necessary promoter, RBS, and Terminator parts, along with the RCF1000 fusion sites. We planned to include eight Level 1 transcriptional unit composite parts in the phagemid (see Figure 3 and Table 2).
Figure 3: SBOL diagrams illustrating the design of two Level 1 transcriptional units, and their component parts. Design of BBa_K4268010 (Level 1 Capsid protein) and BBa_K4268011 (Level 1 Capsid protein with biotin tag) are shown.
Table 2: Design of the remaining Level 1 composite parts required for construction of the phagemid.
| Level 1 composite | Promoter | RBS | CDS | Terminator |
|---|---|---|---|---|
| BBa_K4268014 | BBa_R0085 | BBa_B0034 | Internal core protein BBa_4268014 |
Bba_B0015 |
| BBa_K4268008 | BBa_R0181 | BBa_B0034 | Head-tail connector BBa_4268000 |
Bba_B0015 |
| BBa_K4268012 | BBa_R0181 | BBa_B0034 | Tail tubular protein A BBa_4268004 |
Bba_B0015 |
| BBa_K4268013 | BBa_R0181 | BBa_B0034 | Tail tubular protein A BBa_4268005 |
Bba_B0015 |
| BBa_K4268009 | BBa_R0181 | BBa_B0034 | Capsid assembly protein BBa_4268001 |
Bba_B0015 |
| BBa_K4268015 | BBa_R0183 | BBa_B0034 | Tail fiber-like BBa_4268007 |
Bba_B0015 |
We began performing a Level 1 assembly of the capsid and capsid with biotin tag transcriptional units (TUs). We cloned the promoter, RBS and terminator sequences into pSB1C00, and then assembled the sequences into the Level 1 vector pSB1K03 using Golden Gate assembly. After transformation of the ligation product, we again screened potential positive clones using colony PCR. Results of the colony PCR indicate successful cloning of the Capsid TU into the Level 1 vector (Figure 4). Unfortunately, the assembly of the biotin tagged version of the capsid was not successful. We are continuing to work to clone this part, along with working to assemble the remaining Level 1 TUs and have begun exploring alternative approaches to obtaining the large basic parts that are still needed for Level 0 cloning.
Figure 4: Colony PCR used to confirm Level 1 cloning of capsid protein TUs. The PCR products observed for the Capsid TU colonies matched the expected size of 1211bp. Bands observed for the Capsid with Biotin Tag TU do not correspond with the predicted size of 1271bp.
While we have been unable to progress in the wet lab aspects of the project beyond beginning to clone the Level 1 transcriptional units needed to build our phagemid, we completed the design of the final phagemid. This design will require the assembly of the Level 1 TUs into level 2 multi-transcriptional units (MTU), and then the final assembly of these expression MTUs into the final phagemid.
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 (2,3). 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 elected to take cues from gene order in operons and ordered 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 ise critical to clone the Level 1 parts into the appropriate Level 1 vector. This information is available in the iGEM parts repository under Type IIS assembly (4). In the design of the BBa_K4268017 and BBa_K4268018 phagemids, a “Dummy sequence” BBa_K3425026, designed by Team UofUppsalla in 2020, was included to replace the fourth part in a Type IIS assembly to ensure correct assembly orientation.
Figure 5: SBOL diagrams illustrating the design of the three Level 2 multi-transcriptional units, and their component Level 1 transcriptional units.
Likewise, for final assembly of the MTUs into the S-TIP37 phagemid (Figure 6), orientation and use of the correct level 2 vectors will be important to ensure the assembly corresponds to our preliminary designs. In the design of the BBa_K4268019 and BBa_K4268020, “Dummy sequences” BBa_K3425029 and BBa_K3425030 (Team UofUppsalla, 2020) were included to replace the third and fourth part in a Type IIS assembly to ensure correct assembly orientation.
Figure 6: SBOL diagrams illustrating the design of two S-TIP37 phagemids – one with a biotin-tagged capsid (BBa_K4268020) and an untagged version (BBa_K4268019). Part numbers of the 2 multi-transcriptional units and the dummy parts used are shown.
References
- Kemp, P., Garcia, L. R., & Molineux, I. J. (2005). Changes in bacteriophage T7 virion structure at the initiation of infection. Virology, 340(2), 307–317. https://doi.org/10.1016/j.virol.2005.06.039
- Wells, J. N., Bergendahl, L. T., & Marsh, J. A. (2016). Operon Gene Order Is Optimized for Ordered Protein Complex Assembly. Cell reports, 14(4), 679–685. https://doi.org/10.1016/j.celrep.2015.12.085.
- Lim, H. N., Lee, Y., & Hussein, R. (2011). Fundamental relationship between operon organization and gene expression. Proceedings of the National Academy of Sciences of the United States of America, 108(26), 10626–10631. https://doi.org/10.1073/pnas.1105692108.
- Parts.igem.org. 2022. Help:Standards/Assembly/Type IIS - parts.igem.org. [online] Available at: