Composite

Part:BBa_K2389010:Design

Designed by: Rochelin Dalangin   Group: iGEM17_UAlberta   (2017-10-26)
Revision as of 03:30, 2 November 2017 by Ethanagena (Talk | contribs)


pT8O-LZ


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 633
    Illegal BamHI site found at 1605
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE 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
  • 1000
    COMPATIBLE WITH RFC[1000]


Design

Existing kits of BACTH has T18 and T25 in two different plasmids (Figure 2)

T--UAlberta--DESIGN1.png

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).

T--UAlberta--DESIGN2.png

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