Difference between revisions of "Part:BBa K3114012"
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[[Image:T--Calgary--MoClo1.png|900px|thumb|center|Figure 1. Fusion sites used in the MoClo standard for Golden Gate assembly (Weber et al., 2011).]] | [[Image:T--Calgary--MoClo1.png|900px|thumb|center|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. | + | 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=== | ||
===Sequences and Features=== | ===Sequences and Features=== | ||
Revision as of 08:09, 21 October 2019
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).
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
Sequences and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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