Part:BBa_K3114012

T7 inducible promoter and strong RBS

Usage and Biology

This part is a composite consisting of a T7 inducible promoter (BBa_I719005) and a strong ribosome binding site (BBa_B0030) that has been made to be compatible with the MoClo standard for Golden Gate assembly (Weber et al., 2011).

This part was used to create 12 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)
• Chlorophyll B Reductase (CBR) circuit (BBa_K3114024)
• 7-Hydroxymethyl Chlorophyll A Reductase (7-HCAR) circuit (BBa_K3114025)
• Magnesium Dechelatase Stay Green (SGR) circuit (BBa_K3114026)
• Pheophytinase (PPH) circuit (BBa_K3114027)

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’ promoter fusion sequence included in this part is GGAG, and the 5’UTR 3’ fusion sequence is AATG (Weber et al., 2011).

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

This part was designed in a manner that maintains the proper 8 bp AT-rich sequence between the RBS and the start codon, which is part of the 3’ fusion site.

Characterization

This part has been demonstrated to work in the genetic circuit BBa_K3114022. Cultures were grown to OD₆₀₀=0.4, then induecd with 100mM IPTG. High production levels of 6GIX was achieved as demonstrated by the SDS-PAGE results below.

Figure 3. SDS-PAGE gel showing whole cell lysate (WCL) and Ni-NTA purification fractions (Elutions 1-4) for 6GIX without a signal peptide and an empty vector control (EVC). The marker used is the NEB colour protein standard. The arrow denotes correct band size of 21 kDa for the 6GIX protein.

Sequences and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 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 67
  

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