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pT8O-LZ

BACTH in a Plasmid

First introduced in 1998, the bacterial two-hybrid system (BACTH) based in Escherichia coli facilitated the screening of interactions between two proteins, and provided a simpler, yet powerful, alternative to the well-known yeast two-hybrid technology (Karimova et al.; Battesti and Bouveret). BACTH utilizes the catalytic domain of Bordetella pertussis adenylate cyclase. Here, the two complementary fragments of adenylate cyclase, T18 and T25, are each fused to one of the proteins of interest. Good interaction between the two proteins allows for the reconstitution of the two halves of adenylate cyclase, thus restoring the synthesis of cyclic AMP (cAMP) from ATP (Figure 1). In catabolic operons, such as the lac operon, cyclic AMP binds to the catabolite activator protein (CAP), increasing the affinity of CAP for DNA, and thus transcription through CAP’s interaction with RNA Polymerase.

Sequence and Features

Construct Building and Validation

To facilitate cloning of the redesigned BACTH insert (ordered from IDT), we digested a plasmid containing a fluorescent reporter system (BBa_K611016) using EcoRI/SpeI and ligated our BACTH insert into the resulting vector. Because pT8-O would lead to non-fluorescent colonies, we were able to easily identify the correct colonies from a plate of the transformed ligation product. We then confirmed the sequence of pT8-O using Sanger sequencing.

As a first quick test of our construct, we transformed pT8-O and a plasmid not containing adenylate cyclase (as a negative control) into BTH101, a strain of E. coli that has the lac operon but lacks the adenylate cyclase gene (Δcya-), and plated the transformants onto a plate containing X-gal. As shown in Figure 4, the bacteria transformed with pT8-O appear blue, while the bacteria transformed with the negative control remained white. These results are in line with our expectations, confirming the dimerization of leucine zippers and consequent production of cAMP. We expect bacteria with restored cAMP production, but not bacteria without, to turn blue due to the activation of the lac operon from X-gal being the only carbon source.

For a more quantitative validation of pT8-O, we ran a b-galactosidase assay, as described by Battesti and Bouveret(Battesti and Bouveret), on pT8-O and a negative control. Our results (Figure 5), clearly show that the culture containing the pT8-O plasmid has higher absorbance at OD420 than the negative control, suggesting the presence of active b-galactosidase. These results were also visually confirmed by the culture’s distinctly yellow color in the 96-well plate.

Demonstrating Different Levels of cAMP Production from Different Interaction Strengths

We wanted to demonstrate that different levels of interaction lead to different levels of cAMP production, so based on the results of van Heeckeren and colleagues (van Heeckeren, Sellers and Struhl), we introduced mutations into the leucine zipper sequence of pT8O to generate different variants. We made variants where the leucine zipper attached to T18 was deleted (pT8O-LZ stop; BBa_K2389030), the leucine zipper was mutated to abolish dimerization (pT8O-LZ nda; BBa_K2389020), and the leucine zipper was mutated to impair dimerization (pT8O-LZ sla; BBa_K2389040). Unfortunately, we were not able to intensively test the variant with impaired dimerization (BBa_K2389040); testing was confined to a visual comparison of a liquid culture containing X-gal against another liquid culture of bacteria expressing pT8-O.

Our first assay for measuring relative strengths of cAMP production was the b-galactosidase assay. As seen in Figure 6, the three replicates for pT8-O showed a significant increase in absorbance over the duration of the assay, while the replicates for both pT8O-LZ stop and pT8O-LZ nda remained at baseline level. Figure 7 shows the change in OD420 over the change in time, normalized to the OD600. One-way ANOVA indicates a significance difference in the relative b-galactosidase activity (p < 0.0001). Tukey’s multiple comparisons test reveal significant differences in the b-galactosidase activity of pT8-O vs. pT8O-LZ stop (p < 0.0001) and pT8-O vs. pT8O-LZ nda (p < 0.0001), but no significant difference in the intensities of pT8O-LZ stop vs. pT8O-LZ nda (p > 0.05). Taken together, these results show that changing the strength of the interaction affects the production of cAMP, and consequently, the expression of b-galactosidase from the lac operon.

We found that while the b-galactosidase assay is simple, it still took our team members ~1 hour to prepare and assay these 9 samples, excluding the time it took to make the buffers. To simplify the analysis and minimize the time it takes to get a relative measurement of their strengths, we designed a fluorescent protein-based reporter (BBa_K2389070) and inserted it into pSB1C3 (for submission) and pSB1A3 (for use). We co-transformed each of the three pT8-O variants with this reporter, and measured the fluorescence readout from a sample of their cultures. Using one-way ANOVA, we found that there is a significant difference in the observed fluorescence intensity for each pT8-O variant (Figure 7; p < 0.0001). Tukey’s multiple comparisons test reveal significant differences in the fluorescence intensities of pT8-O vs. pT8O-LZ stop (p < 0.0001) and pT8-O vs. pT8O-LZ nda (p < 0.0001), but no significant difference in the intensities of pT8O-LZ stop vs. pT8O-LZ nda (p > 0.05). These results are consistent with our expected results.

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.