Library · Biology & lab methods
What Microbial Colony Screening & Selection got wrong, from 19 theses
Summary written by a model from the records below: The records describe common failure points encountered when screening and selecting microbial colonies and clones. Researchers frequently observed issues including assay artifacts on solid media, evolutionary circumvention of selection pressures, and poor reproducibility between plate and liquid cultures. These records come from graduate theses, 2021 to 2025, each labeled with its degree where the repository states it, and each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Plus 8 problems the authors left open, listed at the end.
Failure modes, by theses reporting them
Select a mode to jump to its records.
Microbial populations evolve bypass mechanisms that circumvent selective screening circuits
Growth-based selections and continuous evolution systems frequently failed because cells adapted to circuit dynamics or inactivated reporters rather than improving the target activity. In other cases, selective pressures caused severe library diversity bottlenecks or failed to enrich desired phenotypes over controls.
Tried and failed
directed evolution in bacterial selection system applied to genome editing enzymes for mammalian cells. Outcome: did not generalise. Reason: evolved variants optimized propagation by escaping bacterial circuit inhibition rather than improving target mammalian editing activity
Development of Enhanced Base Editors and Prime Editors through Protein Engineering and Directed Evolution · Harvard · PhD
Tried and failed
metabolic detoxifier coexpression in growth selection applied to enzyme directed evolution library screening. Outcome: no signal. Reason: alleviating intermediate toxicity sped growth but failed to alter evolutionary selection pressure away from low-activity variants
Engineering of human enzymes for amino acid depletion therapy in cancer · UT Austin · PhD
Tried and failed
continuous directed evolution using bacteriophage selection circuits applied to multicomponent precision genome editing agents. Outcome: did not generalise. Reason: evolved mutations adapted to phage circuit dynamics by reducing repression rather than improving catalytic activity in target hosts
Enhancing precision genome editing agents through directed evolution and protein engineering · Harvard · PhD
Tried and failed
serial passaging under antibiotic selection pressure applied to heterologous plasmid reporter expression stability. Outcome: unstable. Reason: loss of reporter expression occurred through mechanisms outside coding sequence mutations depending on selection marker
Antibiotic Markers: An Overlooked Design Choice in Synthetic Biology? · UT Austin
Tried and failed
combinatorial transcription factor rewiring via iterative selection applied to bacterial stress tolerance improvement. Outcome: no signal. Reason: iterative chemical selection on a highly diverse combinatorial library failed to enrich superior phenotypes over control
Engineering genetic regulation in Clostridium to improve industrial properties · Imperial · PhD
Tried and failed
pre-screening library variants in an intermediate host applied to synthetic promoter library construction. Reason: selection pressure caused severe diversity bottleneck, yielding broken promoters and sequence deletions
Solid agar matrices and colony biomass interfere with optical and molecular screening assays
Agar components and whole-cell colony material frequently inhibited downstream reactions such as colony PCR or altered DNA melting temperatures. In addition, biosensors and secreted metabolites failed to yield sufficient fluorescence or clearance signals directly on agar plates, and opaque formulations obscured colony visualization.
Considered and rejected
Considered and rejected: Rejected using pour-plating for colony PCR screening due to agar contamination inhibiting PCR and causing false positives.
Establishment of tools for genetic modification of the thermophilic methanogenic archaeon Methanothermobacter thermautotrophicus deltaH · Tübingen · PhD
Considered and rejected
Considered and rejected: Rejected using melting temperatures (Tm) from purified plasmid test qPCRs as a control filter for colony qPCR screening because E. coli colonies alter the Tm.
Towards mapping large-scale deletions in synthetic yeast chromosomes in high-throughput Auf dem Weg zur Kartierung großflächiger Deletionen in synthetischen Hefechromosomen mit hohem Durchsatz · University of Marburg · PhD
Tried and failed
solid medium fluorescence biosensor screening applied to bacterial biosensor strains. Outcome: no signal. Reason: the biosensor strain failed to generate sufficient fluorescence signal on agar plates
High-throughput isolation of anaerobic arsenic-transforming microorganisms · EPFL · PhD
Considered and rejected
Considered and rejected: Rejected Sp-biosensor in favor of Nd-biosensor for isolate screening due to lower sensitivity to MMAs(III), inability to sense DMAs(III), and weak agar signal.
High-throughput isolation of anaerobic arsenic-transforming microorganisms · EPFL · PhD
Considered and rejected
Considered and rejected: Directly testing Pluronic-formulated nanosuspensions on agar plates was rejected because the milky opaque liquid obscured yeast colony counting, requiring testing in DMSO instead
Antifungal Drug Formulation for Vaginal Yeast Infection · Johns Hopkins · Master's
Phenotypes observed on solid agar media fail to translate to liquid culture conditions
Microbial isolates displaying active secretion or clearing zones on agar frequently failed to produce active compounds or degrade substrates in liquid culture. Furthermore, colony replica plating and direct plate streaking resulted in poor survival, low efficiency, or diminished sensor recovery compared to liquid outgrowth.
Considered and rejected
Considered and rejected: Rejected direct colony streaking onto sucrose plates without liquid pre-culture for SacB curing due to poor survival and low efficiency.
Toward optimized genetic transformation techniques for soybean [Glycine max (L.) Merr.] · Iowa State · Master's
Considered and rejected
Considered and rejected: Rejected individually picked colony replica-plating for library curing despite preserving uniform barcode abundance, because total recovered barcode and assigned sensor counts were substantially lower than pooled liquid outgrowth.
Engineering bacterial biosensors to explore the mammalian gut · Imperial · PhD
Tried and failed
screening liquid culture broth for secreted antimicrobial activity applied to bacterial isolates from natural microbiome samples. Outcome: no signal. Reason: most solid-media active isolates failed to produce or excrete active compounds in liquid culture broth
HONEY MICROBIOME AND METABOLOME: A VAST RESERVOIR OF NATURAL ANTIMICROBIALS · Cornell · PhD
Primary clone screening produces false positives from assay artifacts and non-specific binding
Putative hits identified during initial microbial and directed evolution screens repeatedly failed during secondary validation assays. Apparent activity was often caused by copurifying contaminants or non-specific clone binding to assay plastic rather than true target function.
Tried and failed
high-throughput directed evolution screening applied to engineered enzyme variants. Outcome: did not generalise. Reason: apparent screening hits were false positives caused by copurifying contaminants affecting initial normalization and readout
Tried and failed
bioassay screening of microbial isolates applied to antimicrobial and toxicity hit identification. Outcome: did not generalise. Reason: initial phenotypic bioactivity hits failed to reproduce during subsequent follow-up testing
An exploration of chemical cues as mediators of marine predator-prey interactions · Georgia Tech · PhD
Tried and failed
ELISA screening of bacterial expression clones applied to single-domain antibody selection. Reason: Screening selected non-specific plastic-binding clones rather than target-specific binders, generating false positives.
Targeting anti-PD-1 and anti-PD-L1 single-domain antibodies to the tumour microenvironment via matrix metalloproteinase activity · Imperial · PhD
Considered and rejected
Considered and rejected: Rejected ELISA-based screening of bacterial supernatants for phage display candidates due to high rates of false positives from plastic-binding nanobodies, switching to flow cytometry.
Targeting anti-PD-1 and anti-PD-L1 single-domain antibodies to the tumour microenvironment via matrix metalloproteinase activity · Imperial · PhD
Selective plating methods fail to isolate or resolve target microbial strains from mixed samples
Selective agar and migration techniques failed to enrich target microbes because fast-growing bacterial contaminants rapidly outgrew slower colonies. Additionally, selective conditions such as thermal stress or varying antibiotic concentrations lacked the phenotypic discrimination needed to recover target subtypes.
Lost to a baseline
Antibiotic selection (e.g., rifampicin) and semi-solid agar migration methods failed as effective strategies for the isolation/enrichment of termite gut treponemes, leading to the use of membrane filtration combined with deep-agar dilution series.
Phylogeny and metabolism of novel spirochetes from cockroaches and evolutionary origin of reductive acetogenesis in termite gut treponemes · University of Marburg · PhD
Tried and failed
thermal selective screening assay applied to detecting low-abundance cold-tolerant spoilage microbes. Outcome: no signal. Reason: the specific target bacterial subtypes were not recovered under standard laboratory thermal stress conditions
Colony counting and picking exhibit high variability and low repeatability
Quantification based on colony counting demonstrated lower repeatability and high variability, particularly on plates with low colony counts. Automated colony picking also proved inferior to single-cell dispensing for reliable bacterial isolation.
Considered and rejected
Considered and rejected: Rejected using automated colony picker (Qpix2 XT) in favor of a single-cell dispenser (Cytena B.Sight) for bacterial isolation.
Systematic analysis of biomolecular binding reactions using microfluidic systems · EPFL · PhD
Left open by the authors
Problems the authors named and did not get to.
Left open
Perform directed evolution with negative screening on SalR variants to eliminate unintended cross-reactivity with cellobiose. Blocker: Requires a molecular biology wet lab for directed evolution and screening assays
Engineering Synthetic Allosteric Transcription Factors · Georgia Tech · PhD
Left open
Optimize the 96-well conjugation screening protocol to minimize false positives and negatives caused by motile colony lifting in soft agar overlays. Blocker: Requires a wet-lab setup and bacterial culture experiments
Synthetic biology approaches for engineering bacteria as living therapeutics · MIT · PhD
Left open
Implement negative selection schemes in the continuous adenovirus directed evolution platform to evolve biomolecules with enhanced specificity. Blocker: Requires a molecular biology wet lab and mammalian cell culture facilities.
Continuous Directed Evolution in Mammalian Cells · MIT · Doctorate
Left open
Incorporate kinetic selection into directed evolution of hemi-methylated DNA binding domain to enhance epigenotyping assay sensitivity. Blocker: Requires wet lab facilities for yeast surface display, FACS, and directed evolution experiments
Applications of Engineered Proteins in Redox Biology and Biomarker Detection Assay Development · MIT · PhD
Left open
Perform multiple rounds of directed evolution library screening on MIOX from glucose substrate using the developed biosensor. Blocker: Requires a wet lab, engineered bacterial strains, and experimental screening apparatus
Towards biosensor-assisted directed evolution of myo-inositol oxygenase · MIT · PhD
Left open
Clone two gIII-neg construct copies with unique off-target PAMs into the PACE selection system to test multiplexed negative selection stringency. Blocker: Requires wet lab molecular biology, phage culturing, and continuous directed evolution experimental setup
Engineering and evolution of precision genome editing agents · Harvard · PhD
Left open
Conduct a suppressor genetic screen to validate the relationship between Hfq foci dispersal dynamics and bacterial recovery/survival. Blocker: Requires a wet biology laboratory, bacterial culturing, genetic screening, and live-cell microscopy apparatus.
Glutathione contributes to the adaptive response to nitrogen starvation in Escherichia coli · Imperial · PhD
Left open
Apply purifying selection iteratively in continuous evolution experiments until desired enzymatic activity or physical constraints are achieved. Blocker: Requires wet lab facilities, microfluidic droplet sorting apparatus, and continuous directed evolution reagents
Techniques for High-Throughput Directed Enzyme Evolution · Harvard · PhD
Who has worked on this
Authors of the public theses cited on this page, most-cited first.
- Hugo Sallet · EPFL · PhD · 2025
High-throughput isolation of anaerobic arsenic-transforming microorganisms - Elliot James MacLeod · Imperial · PhD · 2024
Targeting anti-PD-1 and anti-PD-L1 single-domain antibodies to the tumour microenvironment via matrix metalloproteinase activity - Xinge Diana Zhang · Harvard · PhD · 2025
Techniques for High-Throughput Directed Enzyme Evolution - Ashley N. Hersey · Georgia Tech · PhD · 2025
Engineering Synthetic Allosteric Transcription Factors - Timon Lindeboom · University of Marburg · PhD · 2025
Towards mapping large-scale deletions in synthetic yeast chromosomes in high-throughput Auf dem Weg zur Kartierung großflächiger Deletionen in synthetischen Hefechromosomen mit hohem Durchsatz - Liang-Chun Chen · Iowa State · Master's · 2025
Toward optimized genetic transformation techniques for soybean [Glycine max (L.) Merr.] - Yulin Song · University of Marburg · PhD · 2025
Phylogeny and metabolism of novel spirochetes from cockroaches and evolutionary origin of reductive acetogenesis in termite gut treponemes - Smriti Pandey · Harvard · PhD · 2024
Enhancing precision genome editing agents through directed evolution and protein engineering - En Ze Linda Zhong-Johnson · MIT · PhD · 2024
Biochemical Analysis of Poly(ethylene terephthalate) Film Degradation Kinetics of Engineered IsPETase Variants - Clare Robinson · Imperial · PhD · 2024
Engineering bacterial biosensors to explore the mammalian gut - Harriet Ellis · Imperial · PhD · 2024
Glutathione contributes to the adaptive response to nitrogen starvation in Escherichia coli - Timothy Lott · Cornell · PhD · 2023
GRAM-NEGATIVE BACTERIA AND MICROBACTERIUM SPP. REPRESENT GREATER PROCESSING CHALLENGES THAN PSYCHROTOLERANT SPORE-FORMING BACTERIA DO FOR EXTENDING THE SHELF-LIFE OF HIGH TEMPERATURE, SHORT TIME (HTST
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