Part:BBa_K3114013

Double terminator wip

Secondary Structure

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Measurement

• [http://openwetware.org/wiki/Cconboy:Terminator_Characterization/Results How these parts were measured]

Usage and Biology

This part consists of the double terminator BBa_B0014 that has been modified to be compatible with the MoClo standard for Golden Gate assembly (Weber et al., 2011).

This part was used to create 8 inducible genetic circuits for high-level production of various proteins using parts from our collection. The circuits are listed below.

• DsbA signal peptide 6GIX circuit (BBa_K3114016)
• MalE signal peptide 6GIX circuit (BBa_K3114017)
• OmpA signal peptide 6GIX circuit (BBa_K3114018)
• PhoA signal peptide 6GIX circuit (BBa_K3114019)
• YcbK signal peptide 6GIX circuit (BBa_K3114020)
• TorA signal peptide 6GIX circuit (BBa_K3114021)
• 6GIX circuit with no signal peptide (BBa_K3114022)
• ModGIX circuit with no signal peptide (BBa_K3114023)

Design

When designing this part and the rest of our collection, we were interested in creating parts that could be used in Golden Gate assembly right out of the distribution kit without the need to first domesticate them in a Golden Gate entry vector. As such, these parts are not compatible with the iGEM Type IIS RFC[1000] assembly standard because we included the BsaI restriction site and MoClo standard fusion site in the part’s sequence.

As per the MoClo standard, the 5’ terminator fusion sequence included in this part is GCTT, and the 3’ terminator fusion sequence is CGCT (Weber et al., 2011).

Figure 1. Fusion sites used in the MoClo standard for Golden Gate assembly (Weber et al., 2011).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 109
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1
  Illegal BsaI.rc site found at 112
  

References

Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PLoS ONE, 6(2). https://doi.org/10.1371/journal.pone.0016765