The proposed constructs that will be generated as a part of project FRaPPe are tabulated below.

BADA SA TABLE KA IMAGE SAYANTANI BANA DENA, GYAN REMIND !!!!

Table 1: Information on proposed FRaPPe BioBricks including sources, existing similar Bricks (which could not be procured this year) and cloning strategy to generate them.

. Construct C4 contains coding sequences for enhanced YFP (Yellow Fluorescent Protein), and Dengue Virus Serotype 1 Non-Structural Protein 5 (TSV08 Strain, GenBank Accession : KR919821.1) under the control of a CMV promoter (Human cytomegalovirus immediate early promoter) and CMV enhancer (Human cytomegalovirus immediate early enhancer). It comes equipped with a high-copy-number ColE1/pMB1/pBR322/pUC origin of replication and selectable marker for Neomycin/Kanamycin resistance (aminoglycoside phosphotransferase from Tn5). It will be created by excising the FRB CDS from Addgene plasmid # 20148 using BamHI at 5’ and MfeI at 3’ to first generate FP tag control 1. This will be followed by cloning the DENV NS-5 CDS (gene synthesized by overlap extension) using HindIII at 5’ and SalI at 3’ into FP tag control 1 to generate FRaPPe C4.

Construct C5 contains coding sequences for enhanced CFP (Cyan Fluorescent Protein), and human Signal Transducer and Activator of Transcription (hSTAT2, GenBank Accession : U18671.1) under the control of a CMV promoter (Human cytomegalovirus immediate early promoter) and CMV enhancer (Human cytomegalovirus immediate early enhancer) in one open reading frame. It comes equipped with a high-copy-number ColE1/pMB1/pBR322/pUC origin of replication and selectable marker for Neomycin/Kanamycin resistance (aminoglycoside phosphotransferase from Tn5). It will be created by excising the FKBP CDS from Addgene plasmid # 20160 using AarI at 5’ and XhoI at 3’ to first generate FP tag control 2. This will be followed by cloning the hSTAT2 CDS (gene synthesized by overlap extension) using XhoI at 5’ and EcoRI at 3’ into FP tag control 2 to generate FRaPPe C5.

Construct C2 contains coding sequences for enhanced YFP (Yellow Fluorescent Protein), and FRB (FKBP-Rapamycin Binding domain of Mammalian Target of Rapamycin) under the control of a CMV promoter (Human cytomegalovirus immediate early promoter) and CMV enhancer (Human cytomegalovirus immediate early enhancer). It comes equipped with a high-copy-number ColE1/pMB1/pBR322/pUC origin of replication and selectable marker for Neomycin/Kanamycin resistance (aminoglycoside phosphotransferase from Tn5). It will be created as per the details mentioned in Table 1.

Construct C1 contains coding sequences for enhanced CFP (Cyan Fluorescent Protein), and FKBP (human FK506 ligand binding protein) under the control of a CMV promoter (Human cytomegalovirus immediate early promoter) and CMV enhancer (Human cytomegalovirus immediate early enhancer) in one open reading frame. It comes equipped with a high-copy-number ColE1/pMB1/pBR322/pUC origin of replication and selectable marker for Neomycin/Kanamycin resistance (aminoglycoside phosphotransferase from Tn5). It will be created as per the details mentioned in Table 1.

Construct T2 contains coding sequences for enhanced YFP (Yellow Fluorescent Protein), Dengue Virus Serotype 1 Non-Structural Protein 5 (TSV08 Strain, GenBank Accession : KR919821.1) and FRB (FKBP-Rapamycin Binding domain of Mammalian Target of Rapamycin) under the control of a CMV promoter (Human cytomegalovirus immediate early promoter) and CMV enhancer (Human cytomegalovirus immediate early enhancer). It comes equipped with a high-copy-number ColE1/pMB1/pBR322/pUC origin of replication and selectable marker for Neomycin/Kanamycin resistance (aminoglycoside phosphotransferase from Tn5). It will be created by cloning the DENV NS-5 CDS (gene synthesized by overlap extension) using HindIII at 5’ and SalI at 3’ into Addgene plasmid # 20148.

Construct T3 contains coding sequences for enhanced CFP (Cyan Fluorescent Protein), human Signal Transducer and Activator of Transcription (hSTAT2, GenBank Accession : U18671.1) and FKBP (human FK506 ligand binding protein) under the control of a CMV promoter (Human cytomegalovirus immediate early promoter) and CMV enhancer (Human cytomegalovirus immediate early enhancer). It comes equipped with a high-copy-number ColE1/pMB1/pBR322/pUC origin of replication and selectable marker for Neomycin/Kanamycin resistance (aminoglycoside phosphotransferase from Tn5). It will be created by cloning the hSTAT2 CDS (gene synthesized by overlap extension) using XhoI at 5’ and EcoRI at 3’ into Addgene plasmid # 20160.

Construct lenti T2 contains coding sequences for enhanced YFP (Yellow Fluorescent Protein), Dengue Virus Serotype 1 Non-Structural Protein 5 (TSV08 Strain, GenBank Accession : KR919821.1) and FRB (FKBP-Rapamycin Binding domain of Mammalian Target of Rapamycin) under the control of a CMV promoter (Human cytomegalovirus immediate early promoter) and CMV enhancer (Human cytomegalovirus immediate early enhancer). It will be created by excising the FRaPPe modules from T2 by double digestion with AgeI and MfeI followed by PCR amplification using BamHI and SalI flanking sites. The lentiviral mammalian expression plasmid will also be digested with BamHI and SalI to release its eGFP insert and clone the FRaPPe T2 construct in its place. This backbone also comes equipped with a high-copy-number ColE1/pMB1/pBR322/pUC origin of replication and selectable marker for Hygromycin resistance for bacterial selection. It will be used for transduction into HEK293 cells.

Construct lenti T3 contains coding sequences for enhanced CFP (Cyan Fluorescent Protein), human Signal Transducer and Activator of Transcription (hSTAT2, GenBank Accession : U18671.1) and FKBP (human FK506 ligand binding protein) under the control of a CMV promoter (Human cytomegalovirus immediate early promoter) and CMV enhancer (Human cytomegalovirus immediate early enhancer). It will be created by excising the FRaPPe modules from T3 by double digestion with NheI and XhoI followed by PCR amplification using BamHI and SalI flanking sites. The lentiviral mammalian expression plasmid will also be digested with BamHI and SalI to release its eGFP insert and clone the FRaPPe T3 construct in its place. This backbone also comes equipped with a high-copy-number ColE1/pMB1/pBR322/pUC origin of replication and selectable marker for Puromycin resistance for bacterial selection. It will be used for transduction into HEK293 cells.

FRaPPe constructs T4 and T5 will contain enhanced variants of CFP and YFP, namely, mTurquoise and cp173 Venus excised from pCKI-PYRATES (optimisation plasmid, for details visit Experiments section). YFP will be excised from construct T2 by digesting with AgeI/KpnI and replaced with cp173 Venus from pCKI, obtained by KpnI/NotI digestion and PCR amplification with Age I/KpnI flanking sites to generate construct T4. CFP will be excised from construct T3 by digesting with AgeI/AarI and replaced with mTurquoise from pCKI, obtained by MfeI/AgeI digestion and PCR amplified with AgeI/AarI sites to generate construct T5. Enhanced FRET sensors will improve stability, FRET efficiency and dynamic range during quantification of iPEP efficiency.

Chemically induced dimerization (CID) has always been a flourishing technique in establishing protein-protein interaction and has found its way in biological systems to facilitate proper binding of proteins in the presence of an inducer. Our project plans to use CID as a complement to a biological reporter system to check the nature of protein-protein binding and thereby make attempts to inhibit any pathogen protein-host protein interaction.

When investigating interactions between two proteins with complementary reporter tags in yeast two-hybrid or split GFP assays, it remains troublesome to discriminate true from false results and challenging to compare the level of interaction across experiments. In this scenario, we use chemically induced dimerization and thereby alleviate the above-mentioned issue. Additionally, upon misfolding, mistargeting, or low expression of the investigated protein(s), a false negative readout of a split-based system cannot be directly distinguished from a true-negative.

Intriguingly, the 12 kDa human FK506 binding protein (FKBP12) and the 100-amino acid domain of the kinase Target of Rapamycin (TOR) known as the FKBP-rapamycin binding domain (FRB) do not directly interact but dimerize in the presence of the chemical rapamycin. This interaction has been exploited for chemically induced activation of e.g. signalling cascades, changes in subcellular localisation and regulation of protein stability in mammalian and yeast systems. The inert nature and the small size of FKRB and FRB domain help in overcoming the problem, and without the presence of the much-required CID effective binding is scarcely attainable for weakly interacting proteins.

Usage of the CID modules in the FRaPPe test construct thus serves many purposes.

• To be able to modulate the strength of the interaction based on Rapamycin dosage. (Dose dependence assay)
• To mimic signalling time scales. Since PPIs regulating cellular processes are highly localised and dynamic, it is difficult to study in experimental systems. In actual biological processes, these are fast, reversible and regulated in space and time (e.g. Phosphorylation reactions, molecular switches of small GTPases). Conventional genetic methods like RNAi or overexpression used to study protein function operate on larger time scales and hence the dynamics cannot be studied immediately. CID helps overcome this problem by enabling the perturbation to occur in a spatio-temporally confined location. It helps translocate the POIs to close proximity in a matter of seconds or minutes, mimicking the signalling timescales. Reversible CID systems also make the process reversible which is an added benefit. (Speed and reversibility)
• To exploit the thermodynamic and kinetic advantages of these systems in yielding robust results for our PPI.
• To enable an in-built positive control. In systems such as yeast two-hybrid or classical split ubiquitin-based PPI analysis (PPI-dependent reconstitution of a split reporter protein, leads to a measurable endpoint signal), positive read-outs are difficult to compare across platforms. In addition, false negatives cannot be distinguished from true negatives. So, since CID enables conditional reconstitution of the split reporter (i.e., FKBP and FRB interact in presence of Rapamycin) rather than based only on the PPI of interest, it acts as an internal control. This system serves as a tool to improve the certainty that employed protein fusions are functional and non-interacting combinations indeed represent true-negatives.
• To ensure the interaction between our POIs in an experimental system, where DENV infection has not been induced or patient cell lines have not been employed.

FRET (Förster or Fluorescence Resonance Energy Transfer) is a technique to assess a wide range of biological activities such as protein-protein interactions, conformational changes, enzymatic processes and so on, all of which involve molecular proximity. These biosensors are highly sensitive to the separation distance between the interacting components (within 1 to 10 nm range), and this in turn dictates many of the factors to be considered while finding appropriate FRET pairs for the biosensor of interest to obtain optimum FRET efficiency.

In living cells expressing proteins fused with FPs, FRET efficiency can be accurately determined using donor fluorescence lifetime or sensitized acceptor emission intensity

FP-based FRET biosensors are preferable over other methods like small molecules due to its inherent advantages.

• They are easy to construct by adding the gene sequence of the respective FPs to the sensing domain by genetic engineering. The other methods would require additional antibody tagging, limiting their versatility.
• TFPs can be made highly specific by using tissue-specific promoters and can be moulded to function in specific subcellular areas of interest.
• Methods such as transfection can be used to easily introduce these constructs into cells in vitro and in vivo, and intracellular FPs have a high stability due to long half-life times.

Usage of the FRET modules in the FRaPPe test construct thus, also serves many purposes.

• As it enables quantification of dynamic interactions, it is sensitive and easy to visualize.
• Can be used for native species in vivo.
• Since no physical interaction is needed between the donor and acceptor fluorophores (non-radiative transfer of energy occurs), FRET is less likely to interfere with the equilibrium of the target pair. Thus, FP‐based FRET can be advantageous for accurately measuring PPI affinities, provided that the PPI brings the donor and acceptor to within the Föster distance (typically ~3–5 nm) [This proximity requirement will be ensured by incorporating the CID system.]
• FRET‐based approaches keep both the quantity and time to a minimum, thereby enabling scale‐up and HTS for affinity assessment of very large numbers of interacting pairs.
• The theoretically equal stoichiometry of the FPs, yields radiometric readouts independent of sensor concentration.

Hence, the composite FRaPPe biobricks will enable rapid screening of protein protein interactions of choice (by replacing our gene blocks with those of the experimenter’s choice) and facilitate screening of inhibitors using fluorescence readouts.