Biosensor libraries harness large classes of binding domains for construction of allosteric transcriptional regulators
- 1. INDIAN INSTITUTE OF TECHNOLOGYROORKEE Biosensor libraries harness large classes of binding domains for construction of allosteric transcriptional regulators Submitted by:- Sourik Dey 18610023 Seminar Presentation (BTN – 613) M.Sc. Biotechnology (IInd Year)
- 2. 2 • The ability of bacteria to sense specific molecules within their environment and trigger metabolic responses in accordance is an invaluable biotechnological resource. • In synthetic biology, a transcriptional genetic sensor is defined as a unit of DNA that confers the ability to respond to a stimulus by controlling the activity of a promoter. Sensor performance is quantified by its response function; in other words, how the concentration of inducer changes the activity of the output promoter. • Despite three decades of research demonstrating that TFs originate from the fusion of individual gene modules, a general method to create functional fusions of two gene domains has remained an elusive holy grail, due to easily broken allosteric interactions between proteins. • To expand the repertoire of biotechnologically relevant sensors, researchers of George Church Lab at Harvard Medical School developed a strategy for the construction and testing of chimeric TF libraries, based on the fusion of highly soluble periplasmic binding proteins (PBPs) with DNA-binding domains (DBDs). Rationale
- 3. 3 Schematic Diagram of Ligand Responsive Transcription Factor Action LigandTranscription Factor TF-Ligand Complex Here, the focus of the group was exclusively on the development of monogenic intracellular sensors, in the form of new TFs capable of detecting small molecules and enabling the host cell to act as a biosensor for that particular ligand. GFP DBD LBD TF producing gene GFP
- 4. 4 Summary of the approach used for generation of novel TFs Juárez, J.F., Lecube-Azpeitia, B., Brown, S.L. et al. Biosensor libraries harness large classes of binding domains for construction of allosteric transcriptional regulators. Nat Commun 9, 3101 (2018)
- 5. 5 DBD & LBD Combinations Used 1. The novel TFs generated here are based on the fusion of DBDs to LBDs that were not part of preexisting regulators, but rather independent proteins with the ability to bind soluble small molecules. DBD and LBD were connected through a series of linkers (LNKs). 2. All TFs resulting from this combination of domains were functionally tested with benzoate, the inducer molecule specifically recognized by a group of LBDs included within the library. Benzoate was selected for this proof of concept work due to its environmental relevance and Escherichia coli is unable to degrade benzoate and it is relatively nontoxic, allowing for its direct addition to culture media.
- 6. 6 Construct Design For systematic construction of fusion TFs the group generated libraries under the following two degrees of freedom:- (a)15 DBDs sourced from bacterial transcriptional repressors with a common architecture and known operator sequences (b)15 LBDs from PBPs associated with ATP-binding cassette transporters.
- 7. 7 Different Classes of Linkers assembled by eLCR The Enhanced ligase chain reaction (eLCR) is an amplification process that differs from PCR in that it involves a thermostable ligase to join two probes or other molecules together which can then be amplified by standard PCR cycling.
- 8. 8 Functional Screening of Inducible Transcription Factors Functional screening was based on two-step enrichment:- • First step (Negative Sorting) - The population was sorted based on GFP expression (by the reporter promoters) to enrich cells in which a pairing between the DBD of the chimera and the operator boxes had formed (GFP negative). In such cells, the chimeric TF binds to the reporter promoter and represses the expression of GFP. Accordingly, this allowed for functional screening by negative selection to recover a sorted population enriched in properly paired chimera-reporter plasmids. • Second step (Positive Sorting) - Bacteria recovered by negative selection were cultured in the presence of the inducer molecule benzoate, followed by sorting for positive expression of GFP. To be recovered in this step, a bacteria that expressed a functional chimera required: (a) an LBD that could bind benzoate and (b) a subsequent conformational change upon binding that could alter the allostery of the repressor, forcing it to unbind from its DNA operator within the reporter promoter. • In cells where these two events occurred, RNA polymerase σ70 could recognize the unrepressed −10/−35 boxes of the promoter and transcribe GFP, leading to cellular fluorescence.
- 9. 9 FACS Enrichment Technique Juárez, J.F., Lecube-Azpeitia, B., Brown, S.L. et al. Biosensor libraries harness large classes of binding domains for construction of allosteric transcriptional regulators. Nat Commun 9, 3101 (2018)
- 10. 10 Results • Over 100 chimeras showed enrichment in the screening assay, and 2 of these were selected for further characterization owing to their unique features. • First Chimera - CbnR-ABE44898-OD (ChTFBz01), from the AYC Lic-Ch-OD library, because it carried a Ct LacI OD and its LBD (ABE44898) was similar to RPA0668: a Rhodopseudomonas palustris protein whose ability to bind benzoate has already been established. • Second Chimera - LmrR-BzdB1_nSP (ChTFBz02), from the AYC Lib-Ch-END library, as it contained a LBD originally identified as a PBP associated to a cluster responsible for benzoate catabolism. • ChTFBz01 and ChTFBz02 were recloned to pCKTRBS-OD and pCKTRBS, respectively, downstream of the tet-O site and can be induced with aTc capable of removing the TetR repressor. Subsequently, E. coli strains harboring each were transformed with the appropriate reporter vectors. • The resulting strains AYC ChTFBz01 and AYC ChTFBz02 allowed them to assess the ability of CbnRABE44898-OD and LmrR-BzdB1_nSP to repress the expression of GFP and be derepressed by benzoate.
- 11. 11 Graphical Representation of Inducer Dependent Promoter Activity 1. Promoter activity is measured as fold of the relative fluorescence (fluorescence in arbitrary units/OD600) of the strains grown under inducing conditions (cultures supplemented with aTc and benzoate; blue violin plots) or repressing conditions (cultures supplemented with aTc; red violin plots) compared to the basal expression of the promoter (no aTc and no benzoate). 2. It can be appreciated how in order to maintain the basal activity (fold =1) the addition of benzoate to the culture medium is necessary when the chimeras are expressed by the addition of aTc. Juárez, J.F., Lecube-Azpeitia, B., Brown, S.L. et al. Biosensor libraries harness large classes of binding domains for construction of allosteric transcriptional regulators. Nat Commun 9, 3101 (2018)
- 12. 12 Conclusion and Future Directions • The novel sensor proteins developed in this work expand the limited collection of available transcriptional repressors that can be used as biosensors in the degradation of lignin. • Beyond their immediate usability to tackle this biotechnological problem, they represent an important milestone for the construction of synthetic TFs on demand, as the pipeline can be readily applied to create many other custom-made chimeric TFs. • The combination of the LBDs and DBDs can give us an immense repertoire of TFs exclusively tailored for different xenobiotics that can be used as inducible elements controlling the downstream expression of other genes.
- 13. 13 References • Juárez, J.F., Lecube-Azpeitia, B., Brown, S.L. et al. Biosensor libraries harness large classes of binding domains for construction of allosteric transcriptional regulators. Nat Commun 9, 3101 (2018). • Fukami-Kobayashi, K., Tateno, Y. & Nishikawa, K. Parallel evolution of ligand specificity between LacI/GalR family repressors and periplasmic sugar-binding proteins. Mol. Biol. Evol. 20, 267–277 (2003). • De Kok, S. et al. Rapid and reliable DNA assembly via ligase cycling reaction., ACS Synth. Biol. 3, 97–106 (2014). • Collier, L. S., Gaines, G. L. 3rd & Neidle, E. L. Regulation of benzoate degradation in Acinetobactersp. strain ADP1 by BenM, a LysR-type transcriptional activator. J. Bacteriol. 180, 2493–2501 (1998).