Team:BostonU/FusionProteins

From 2014.igem.org

• TEAM
• PROJECT
  • Overview
  • Priority Encoder
  • MoClo Assembly Method
  • Low Copy Backbones
  • Tandem Promoters
  • Fusion Proteins
  • Repressor Proteins
  • Multiplexing
  • BioDesign Automation Tools
  • Future Work
• ACHIEVEMENTS
  • Data Collected
  • Parts Submitted
  • Chimera Workflow
  • Chimera Example
  • Medal Fulfillment
• NOTEBOOK
  • Training
  • Protocols
  • Backbones
  • Tandem Promoters and Repressors
  • Fusion Proteins
• CONSIDERATIONS
  • Collaborations
  • Measurement Track
  • Safety
  • Interlab Study
  • NEGEM
  • Policy and Practices
• ACKNOWLEDGEMENTS

Fusion Proteins

Why Fusion Proteins? Fusion Proteins are fused coding sequences that allow us to use one transcriptional unit (with a repressor and reporter protein fused together) rather than two units (one with the repressor and the other with the reporter protein, whose expression depends on the expresser of the repressor). Fusion Proteins play a very important part in Project Chimera. In addition to helping us reduce the total transcriptional units used, they also allow for greater expression of tandem coding sequences. If we look at constructs that have repeating sequences of ribosome binding sites and reporter proteins in one transcriptional unit, a peculiar effect can be observed. When ribosomes bind to the genes sequentially, the ribosome that binds before might block the next ribosome from binding to the mRNA and translating the gene. So, the later genes are expressed less than the genes before. This problem is solved with fused proteins as only one ribosome will then be required to translate the entire sequence, eliminating any possible problems during translation. Assembly To make fusion proteins, we used the Modular Cloning method that we have used for most digestion-ligation reactions. First, we added a new MoClo fusion site (I - TCTA) to the genes (at the end of repressors and before the reporter proteins). The fusion site, I (TCTA) was then added to another 2-nucleotide sequence (GA) to make the XbaI site. This was done to allow the two proteins made by the two gene to split up after translation. Phusion Polymerase Chain reaction was performed on the basic MoClo Level 0 parts using primers designed based on the fusion sites. We ended up with repressors with TCTAGA sequence at their ends and reporter proteins with the same sequence at the start. These can be treated as standard Level 0 parts which can then be assembled and tested using MoClo. Testing It is important that the fusion proteins made aren’t drastically inferior to the individual action of the repressor or the fusion proteins. In order to test this, the following Level 2 constructs were assembled using MoClo - J00-C12_AE - R10-C40-E40m_EF - R40-E10_FG J00-C12_AE - R10-C40-E30_EF - R40-E10_FG J00-C12_AE - R10-C80-E40m_EF - I13-E10_FG J00-C12_AE - R10-C80-E30_EF - I13-E10_FG Here onwards, all constructs made were to compare with those above. Simply put, all 6 Level 2s below were used a controls. J00-C12_AE - R10-C80_EF - I13-E10_FG J00-C12_AE - R10-E30_EF - I13-E10_FG J00-C12_AE - R10-E40m_EF - I13-E10_FG J00-C12_AE - R10-C40_EF - R40-E10_FG J00-C12_AE - R10-E30_EF - R40-E10_FG J00-C12_AE - R10-E40m_EF - R40-E10_FG * All constructs contain BCD2_BC as 5' UTRs and B0015 as terminators. Detailed progress on the construction of fusion proteins can be found in the Fusion Proteins notebook.

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