Motivation: Temperature-sensitive (TS) plasmids are those plasmids that can replicate till a permissible temperature (say 30°C), but they lose their ability to replicate at higher temperature (say 35°C), and thus, the plasmid is lost by the cell in successive replication cycles. TS plasmids are important in synthetic biology as they give the ability to control the burden on any cell by altering the plasmid replication as a function of temperature. They can be used in genetic engineering and manipulation as delivery vectors, for inducible replication by supplying protein for transient expression and various other features. Once the plasmid has done the work and has served the purpose, it becomes a burden to the cell, and thus, to reduce the burden, it is important to make the cell plasmid free. The TS plasmid can be cured by raising the temperature.

Work: The goal of this project was to design and construct a temperature-sensitive plasmid that would later contain genes for recombineering (genome engineering). The design of this TS plasmid involved protein engineering steps: predicting the TS mutation, predicting the 3D structure of the replication protein, performing docking with the origin of replication and then proving these predictions by experiments: cloning and screening for the TS phenotype.

Publication: Deedwania A, Karmakar S, Kumar V, Shefrin S, Sundar D, Srivastava P. Construction and characterization of a temperature-sensitive pRC4 replicon for Rhodococcus and Gordonia. Gene. 2023 Nov 15:147990. doi: 10.1016/j.gene.2023.147990. Epub ahead of print. PMID: 37977321. Link to the paper

Motivation: Cell fusion is an important process for various cellular processes including cell-cell communication, drug delivery, cell transfection and other therapeutic processes. For synthetic cells, designing and controlling spatial and temporal biological reactions have been a challenging process. Targeted vesicle fusion has been shown as a promising approach to control interactions between compartments selectively. Membrane fusion is also important to initiate protein synthesis in synthetic cells. Thus, modeling and designing a biomolecular circuit is an important step.

Work: I designed, modeled and simulated a biomolecular subsystem that would export single-stranded DNA (ssDNA) encapsulated inside a synthetic cell (vesicle) and aid the linker-induced fusion of two DNA-tethered synthetic cells that are externally embedded with the complementary strand of the exported ssDNA. Upon the formation of double-stranded DNA, the two cells would fuse. The CRN models were made using BioCRNpyler, compartmentalization was introduced through subSBML and simulations were done using Bioscrape.
An example of the designed subsystem is- The vesicle is encapsulated with cell extract, linker ssDNA and plasmid that can express aHL protein. The input signal, aTc in the external environment will induce the expression of the gene, producing aHL. The protein binds to the membrane and forms a channel through which ssDNA can be exported. This exported ssDNA acts as linker which helps in the controlled fusion of vesicles. This controlled fusion can help us initiate protein expression in other vesicles. For a simple model to initiate protein expression, the liposome population 1 is encapsulated with gene expression of exsA, induced in presence of aTc (present in the external environment). Liposome population 2 is encapsulated with a gene which expresses GFP, which is activated only in presence of exsA, so there is no protein in this liposome as exsA is being expressed in liposome population 1. These are DNA-tethered liposomes. When the exported linker DNA reacts with its complementary strand, it initiates fusion of two populations of liposome, thus exsA can induce the expression of GFP in the fused cell.

The code and models are available at my GitHub repository.

Motivation: RNA Thermometers (RNATs) are temperature-sensitive RNA molecules that can control protein expression. RNATs alter their secondary structure in response to temperature fluctuations. They are present in the 5’ UTR region of mRNA sequence. As temperature increases, the hydrogen bonds break/ melt and thus, expose the regions like ribosome binding sites that affect translation, and permit translation. This structural transition can depend on either the heat shock or cold shock response resulting in either exposing or hiding of RBS. Thus, it was important to understand the molecular mechanism or the structural transition that takes place by temperature change in some naturally occurring RNATs.

Work: I studied the temperature dependence of protein expression of naturally occurring RNATs like rpoH, cspA and agsA. The RNATs sequence was also used to analyze the expression and melt profile via the software tool NUPACK. I also constructed a library of synthetic RNA thermometers expected to give a better melt and expression profile. A single base change was made in the natural RNATs, and the sequence was again analyzed; the ones giving better melt profile were synthesized and tested. .

Project poster is available at this link.

Motivation: Lead poisoning is a catastrophe affecting millions of people around the world. Having a biosensor that could sense lead and also recover it from water could solve this major problem. Previously developed whole-cell lead biosensors cater to a limited sample type i.e. soil samples. Only a few studies have expanded the sample type to wastewater samples, which is one of the main focuses of our biosensor. Whole-cell lead biosensors are intensity-based. Also, industries use various physicochemical methods like chemical precipitation etc for lead removal from industrial wastewater, which brings down the lead concentration from 200-500 mg/L to 0.4 mg/L, but is still ten-fold more than the permissible limit and resulting in poor recovery efficiency beyond this concentration.

Work: We made a system that would assess and monitor lead contamination, and also adsorb lead from water samples and recover it for viable commercial use. We intend to develop a frequency-based biosensor that provides many advantages over intensity-based biosensors like robustness from environmental factors, and also developed a new recovery system for the same using cell surface adsorbtion. A lead binding protein would be expressed on the cell surface that would capture lead from the solution, thus recovering lead even at lower concentrations.

Role: Advisor and mentor to the iGEM IIT Delhi team. Guided in ideation of the project and mentored, taught undergraduate students in the experimental lab and modeling techniques. .

Won the gold medal at the iGEM 2022 competition. The work is available at the iGEM wiki.