Part:BBa_K2389010:Design
pT8O-LZ
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 633
Illegal BamHI site found at 1605 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 141
Illegal NgoMIV site found at 551
Illegal AgeI site found at 357
Illegal AgeI site found at 609 - 1000COMPATIBLE WITH RFC[1000]
Design
Existing kits of BACTH has T18 and T25 in two different plasmids (Figure 2)
Because our RISE system involves transforming another plasmid carrying the genes required to produce gas vesicles (BBa_K2389060) and we know that transformation efficiency suffers from multiple plasmids, we wanted to streamline the BACTH system and combined both T18 and T25 subunits in one plasmid, which we refer to as pT8-O (Figure 3).
To facilitate its use, pT8-O has the following features:
1. Constitutive promoter
The BACTH kit Team UAlberta ordered had both T18 and T25 subunits under the control of the lac promoter, which allowed for inducible expression. However, we wanted to ensure that expression of the two subunits, and the proteins of interest, is not the limiting factor in the production of gas vesicles. Therefore, we replaced the lac promoter with the strongest constitutive promoter in the Anderson promoter collection (BBa_J23100).
2.Strong RBS
When designing this construct, we found a paper by Chen et al. that determined the optimal spacing between the Shine-Dalgarno sequence and the initiation codon in E. coli, and decided to use their reported RBS sequence (Chen et al.).
3. Flexible linkers
We know that orientation matters for protein interaction. Thus, we decided to include flexible linkers between the adenylate cyclase subunits and the proteins of interest to enable free movement of the proteins of interest.
4. Excisable Leucine zippers
Commonly found in DNA-binding motifs, where they serve as a dimerization domain, leucine zippers are alpha helices with a leucine zipper at every seventh residue for eight turns(van Heeckeren, Sellers and Struhl). The commercial kit for BACTH uses leucine zippers as a positive control (possibly also due to their small size of ~100 bp), and we decided to also incorporate the leucine zippers into pT8-O. Flanking the leucine zippers are two unique pairs of restriction enzymes (BglII/KpnI and HindIII/BamHI) so that researchers can work with one subunit without affecting the other subunit.
5. Codon-optimized coding regions
We codon-optimized the coding sequence for T18, T25 and the leucine zippers to increase expression levels and improve the translational efficiency.
Source
Bordetella pertussis
References
Battesti, Aurélia, and Emmanuelle Bouveret. "The Bacterial Two-Hybrid System Based on Adenylate Cyclase Reconstitution in Escherichia Coli." Methods 58.4 (2012): 325-34. Print.
Chen, Hongyun, et al. "Determination of the Optimal Aligned Spacing between the Shine–Dalgarno Sequence and the Translation Initiation Codon of Escherichia Coli M Rnas." Nucleic acids research 22.23 (1994): 4953-57. Print.
Karimova, Gouzel, et al. "A Bacterial Two-Hybrid System Based on a Reconstituted Signal Transduction Pathway." Proceedings of the National Academy of Sciences 95.10 (1998): 5752-56. Print.
van Heeckeren, W. J., J. W. Sellers, and K. Struhl. "Role of the Conserved Leucines in the Leucine Zipper Dimerization Motif of Yeast Gcn4." Nucleic Acids Research 20.14 (1992): 3721-24. Print.