About
Researcher with strong biochemistry, molecular biology and interpersonal skills. Passionate about metabolism, metabolic engineering and protein biochemistry. My prior experience with multiple model organisms and training in a broad spectrum of biochemistry and molecular biology techniques would be an asset in executing projects on time and in training individuals. Believer in perpetual learning to excel in science and look forward to such an opportunity.
Professional Experience
September 2021 - present
Postdoctoral research associate,
University of Kent, UK
Antimicrobial resistance is a major health concern (PMID: 34737424) and an enormous amount of
time and effort is required for the discovery of a new antibiotic. The antibiotics are mainly produced
by a group of soil-dwelling bacteria called actinomycetes, which have gene clusters encoding large
modular proteins referred to as megasynthetases. Understanding how these biosynthetic gene
clusters get activated and learning details of regulation involved in their transcription and
translation is critical to making advancements in antibiotic discovery. My work makes use of
luciferase and fluorescence reporters and a cell-free system of a model actinomycete, Streptomyces
venezuelae (PMID: 33497199) to understand the kinetic aspects of megasynthetase transcription
and translation. I am carrying out extensive optimization of the cell-free system to produce a
sufficient quantity of candidate megasynthetases, as some of them can be as big as the ribosome
itself. I am also trying to understand the activation of cryptic gene clusters in vivo using CRISPR
Cas9 to generate transcriptional and translational reporter fusions. Another key aspect of my
research is to show the applicability of cell-free systems to express multienzyme pathways that are
challenging in cell-based systems. As cell-free reactions are carried out in small volumes in a high-
throughput manner, they can serve as a means for rapid prototyping of metabolic pathway
deployment in non-model organisms without the need for gene manipulation (PMID: 31780816).
November 2019 - February 2021
Scientist I, String Bio Pvt. Ltd., Bangalore
I was an active member in multiple cross-functional teams involved in various projects and also the technical lead of one of the teams. I have optimized assays to determine promoter strength using whole bacterial cells in a 96-well format, established a microscopy-based quantitative tool for determining the composition of a mixed bacterial culture, and developed colorimetric method for estimation of the live-dead percentage of bacterial cells. I also generated a gene deletion methanotroph strain and also worked towards recombinant peptide expression and purification from E. coli system. Additionally, I was also involved in the preparation of the manuscript, project charter and preparing content for submission of the Invention Discovery Form (IDF).
What I’ve Learned
Skills/Expertise/Experience
Molecular Biology
Restriction-digestion based cloning
Ligase-independent assembly cloning (AQUA cloning - Advanced QUick Assembly cloning)
Cloning using In-fusion assembly kit, Golden gate assembly and Gibson assembly
Guide RNA cloning
Cloning by employing phage derived lambda red-recombinase in compatible E. coli strains, and in vitro gateway LR clonase method
Site-directed mutagenesis
Model organisms and genetic engineering
Handled and performed genetic manipulation in various model systems - E. coli, Saccharomyces cerevisiae, Pichia pastoris, Plasmodium falciparum, Plasmodium berghei, Streptomyces venezuelae and the methanotroph Methylococcus capsulatus
Generated knockout, conditional degron, epitope-tagged and transcriptional fusion reporter strains in Methylococcus capsulatus, Saccharomyces cerevisiae, Plasmodium berghei and Streptomyces venezuelae.
Biochemical techniques
Recombinant protein expression and purification (by ammonium sulphate precipitation, Ni-NTA-chromatography, ion-exchange and size-exclusion chromatography)
Western and Southern blotting
Antibody generation and dot blot
Enzyme characterization by continuous and end-point measurement assays as well as in vivo functional complementation assay
Have ample exposure and hands-on experience in CD, fluorescence spectroscopy and microscopy, pre-steady-state kinetics, and mass spectrometry
Performing retro-orbital injections in mice, and blood smear preparations from tail snips.
Cell viability estimation by colorimetry
Cell-free systems
Expertise in cell-free transcription translation and production of proteins using E. coli and Streptomyces venezuelae cell-free system.
Writing and software skills
Data compilation, writing and editing for preparation of high-quality manuscripts
Preparation of project charter and invention discovery forms
ImageJ, PyMOL, GraphPad Prism, MS Office, LaTeX (using LyX)
Publications
A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems
Nagappa LK, Wakana Sato, Farzana Alam, Kameshwari Chengan, Christopher Mark Smales, Tobias von der Haar, Karen Marie Polizzi, Kate Adamala and Simon J Moore. Frontiers in Bioengineering and Biotechnology, 2022
My Volunteer Background
April-May 2019
Student Mentoring Programme
Taught botany for pre-university students from underprivileged background.
2007-2010
National Service Scheme
Volunteered in two village camps spreading awareness about cleanliness, problems of child marriage and the importance of education
Let’s Connect
#1230, 10th main road, 11th cross, 3rd stage, Gokulam, Mysore - 570002
knlakshmeeshaattherategmaildotcom
