Part:BBa_K2109104:Design
We based our two hybrid reporter on the construction based on the BacterioMatch II system, with the following construction:
The complete bacterial-two-hybrid system is composed of three parts:
(1) the lambda cI DNA-binding protein that has a C-terminal fusion to the bait protein (single domain antibody, in our case);
(2) Bacterial RNA polymerase alpha subunit that is C-terminally fused to the prey (target antigen) protein; and
(3) A reporter cassette that confers antibiotic resistance or fluorescence.
Our bacterial two hybrid reporter works on the basis of having lambda cI protein binding site (e.g. OR1) upstream of a weak constitutive promoter (BBa_J23113) that drives transcription of the reporter gene. We decided to employ a fluorescent reporter gene (mRFP1) since this allows for quantitative comparison of mRFP fluorescence levels as opposed to antibiotic markers that require laborious titration of antibiotic to select positive for clones (indicating binding between bait and prey).
Activation of reporter gene expression is stimulated by the interaction of bait (single-domain antibody) and prey (target protein) polypeptides. This in turn brings bacterial RNA polymerase to the weak constitutive promoter via protein-protein interactions, beginning with binding of lambda cI at the operator region OR1 and positioning of RNA polymerase at the promoter via binding of the bait and prey. Fluorescence intensity may be semi-quantitatively correlated to the strength of binding between a bait and prey in the two hybrid system.
We initially envisioned the reporter to be integrated into the bacterial chromosome, which would obviate the problem of plasmid copy number effects for reporter expression during the assay (i.e. noise due to cell-to-cell variation in plasmid copy number). Instead, for the sake of simplicity and ease of manipulation of the reporter (for optimization, etc.) we decided to place the reporter on the same low-medium copy plasmid vector (pSB3K3) as the target-RNA polymerase alpha fusion-expression. A secondary benefit of placing the reporter on a multi-copy plasmid is a relative increase in the sensitivity of the assay due to increased reporter signal (i.e. more copies of reporter equates to more overall reporter fluorescence). However, to correct for plasmid copy number variation, we also introduced a second fluorescent protein on the reporter, mTagBFP under the control of a strong constitutive promoter. The second mTagBFP reporter has essentially no spectral overlap with the first reporter, mRFP1 and provides a tangible reference to compare the mRFP1 signal between cell with different plasmid copy numbers. Thus, during an experiment, the mRFP1 fluorescence will be normalized to that of mTagBFP.
When designing the binding site for lambda cI protein, we opted to employ solely OR1. While there are several binding sites for lambda cI in the phage genome (e.g. OR1-3), OR1 binds the lambda cI dimer the most tightly (low nanomolar dissociation constant) and we reasoned that the single site would be sufficient for efficient binding of the lambda cI-prey (sdAb) protein. We recognized that the combination of OR1 and OR2 results in a higher overall affinity, however these two operator regions overlap a strong transcription promoter and in absence of lambda cI we would observe strong activation of the mRFP1 reporter.
Optimal positioning of the two bait-prey fusion proteins for maximal reporter activation is affected by at least two variables:
(1) the overall separation of the lambda cI and RNA polylmerase moieties as determined by the bulk of the two interacting bait-prey polypeptides; and
(2) the linker distance that separates the OR1 and weak promoter sites.
For our design, we first tried to choose and optimal OR1 – promoter linker distance by considering the approximate length of the bound fusion proteins (20 to 100 aa as spheres) and the fact that a standard DNA helix has ~10 bp per turn. We decided to use a 40 bp linker as this corresponds to ~4 helical turns with the binding site for lambda cI and RNA Polymerase being on the same side of the double helix. Because we were unable to assemble the complete bacterial-2-hybrid system, we were unfortunately unable to evaluate whether our selected cI-promoter distance is optimal and how this is affected by target antigens of differing sizes.
Fluorescent Bacterial-2-Hybrid Reporter Cassette
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 130
Illegal NheI site found at 153
Illegal NheI site found at 1822 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 84
Illegal XhoI site found at 118 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
mTagBFP is encoded on the opposite (antisense) strand and is followed by a strong bidirectional transcription terminator.
Source
This part was synthesized by Integrated DNA Technologies (IDT) as a gBlock and subsequently subcloned into the pJET1.2 cloning vector prior to BioBrick cloning into pSB3K3 and pSB1C3.
===References===