Library · Biology & lab methods
What Genetic Circuit & Plasmid Construction got wrong, from 65 theses
Summary written by a model from the records below: The records describe engineering challenges in constructing synthetic genetic circuits and propagating plasmid vectors across diverse host systems. Circuit failure commonly stemmed from basal expression leakiness, severe metabolic burden, unintended molecular crosstalk, promoter deficiencies, and plasmid instability. These records come from graduate theses, 2021 to 2026, 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 9 problems the authors left open, listed at the end.
Failure modes, by theses reporting them
Select a mode to jump to its records.
Basal promoter leakiness and unintended off state expression compromise genetic switch fidelity
Incomplete transcriptional repression and cis proximity between regulatory elements resulted in high baseline expression across reporters and logic gates. This persistent leakiness reduced dynamic ranges, flipped bistable switches prematurely, and permitted growth without selective induction.
Tried and failed
compact bidirectional promoter design with shared operators applied to multi-output synthetic gene circuits. Reason: operator placement too close to core promoter elements caused off-target regulation and high baseline expression leakiness
Advancing Genetic Circuit Design with Prediction, Reversible Logic, and Thermal Induction · Georgia Tech · PhD
Tried and failed
minimal promoter reporter in synthetic gene circuits applied to measuring synthetic transcription factor activation. Reason: minimal CMV promoter exhibited high basal expression causing reporter signal saturation above detection limits
Drug-Responsive Regulation and Comparison of Engineered Transcription Factors Containing Different Activation Domains · Harvard · Master's
Considered and rejected
Considered and rejected: Rejected combining transcription factor and reporter into a single plasmid because cis-enhancer elements caused ligand-independent constitutive activation
Drug-Responsive Regulation and Comparison of Engineered Transcription Factors Containing Different Activation Domains · Harvard · Master's
Considered and rejected
Considered and rejected: Cre-ERT2 knock-in systems rejected for tumor initiation due to promoter leakiness and complex breeding schemes.
TUMOR SUPPRESSOR GENOTYPE INFLUENCES THE EXTENT AND MODE OF IMMUNOSURVEILLANCE IN LUNG CANCER · Penn · PhD
Considered and rejected
Considered and rejected: hSyn1 promoter for filler construct rejected due to non-sparse labeling and background leak; replaced with inducible ihSyn1 featuring tetO binding sites.
Mapping Memory Encoding and Recall Using Translation-Dependent Reporters of Synaptic Potentiation in Physiological and Pathological Conditions · Scuola Normale Superiore · PhD
Tried and failed
tuning expression in two-state autocatalytic bistable switch applied to extending genetic switch memory duration. Outcome: unstable. Reason: basal promoter leakiness flips the switch state prematurely regardless of expression tuning
Design of a robust ratiometric sensor and a long-term memory genetic toggle switch · MIT · PhD
Tried and failed
split-inhibitor strand displacement logic gates applied to in vitro transcriptional NAND circuits. Reason: unprotected single-stranded promoter regions caused high baseline leak and incomplete repression across off states
Exploring the design principles of orthgonal transcription control systems · UT Austin · PhD
Tried and failed
uninsulated inducible promoter within a mobile genetic element applied to titratable genomic expression control. Reason: high baseline expression caused by read-through from uninsulated flanking sequence
Tried and failed
promoter-based endogenous transcription factor biosensor applied to detecting cellular p53 activity. Reason: low on-state induction coupled with high basal leaky expression in target-deficient cells
Synthetic Genetic Circuits for Cancer Immunotherapy · Harvard · Master's
Tried and failed
Single plasmid containing both regulator and reporter applied to inducible mammalian gene expression systems. Reason: Cis-proximity of regulator and reporter elements caused high basal expression in the uninduced state
Drug-Responsive Regulation and Comparison of Engineered Transcription Factors Containing Different Activation Domains · Harvard · Master's
Tried and failed
recombinase-based toggle switch without positive feedback applied to long-term cellular memory storage. Outcome: unstable. Reason: basal promoter leakiness causes unintended inversion and memory loss over time
Design of a robust ratiometric sensor and a long-term memory genetic toggle switch · MIT · PhD
Tried and failed
conditional metabolic selection using repressed promoter reporter applied to continuous directed evolution of transcription factors. Outcome: no signal. Reason: leaky basal promoter expression in the repressed state was sufficient to permit growth without selection
Development of a targeted mutator system for continuous directed evolution in vivo · Imperial · PhD
Considered and rejected
Considered and rejected: Initial simple split inhibitor NAND gate design was rejected due to 35% leaky OFF-state transcription, replaced by a remote toehold design with a 6 nt spacer and 16 nt promoter overlap
Exploring the design principles of orthgonal transcription control systems · UT Austin · PhD
Considered and rejected
Considered and rejected: Inclusion of the RiboJ genetic insulator in initial reporter plasmids was abandoned because increased basal expression ('leakiness') degraded dynamic range across multiple repressors.
DEVELOPING A TRANSCRIPTIONAL PROGRAMMING EDIFICE USING SYSTEMS OF ENGINEERED TRANSCRIPTION FACTORS · Georgia Tech · PhD
Considered and rejected
Considered and rejected: Rejected inversion-based loxP reporters (flipping promoter or RBS-mCherry) due to high basal leakiness and lower total activation.
Light-inducible tools for control of bacterial gene expression and antibiotic resistance · Boston University · PhD
Considered and rejected
Considered and rejected: Pfdx-RBE promoter-riboswitch cassette for Cas9 control was rejected due to baseline leakiness in the OFF state.
Development of Genetic Tools for the Chemolithoautotroph C. metallidurans CH34: Harnessing Synthetic Biology to Expand Knowledge of Extracellular Electron Transfer · Nottingham · PhD
Considered and rejected
Considered and rejected: Rejected single-operator promoter architectures in Bacteroides due to high basal leakiness and low dynamic range at single genomic copy
Genetic circuit design platforms for engineering living therapeutics · Georgia Tech · PhD
Truncated motifs and incompatible promoter architectures lead to insufficient transcription
Synthetic and hybrid promoters frequently lacked essential core sequence elements or binding sites necessary to drive detectable expression. Cross-species incompatibilities and epigenetic silencing mechanisms further prevented regulatory proteins from activating downstream targets.
Tried and failed
tandem operator promoter engineering applied to synthetic eukaryotic transcription factor reporter. Reason: promoter architecture resulted in insufficient maximum expression levels for functional biosensing
Mapping Alternate Allosteric Communication of LacI Anti-Repressors and Their Application in Saccharomyces cerevisiae - Laying the Foundation for Designing Synthetic Transcription F · Georgia Tech · PhD
Tried and failed
synthetic promoters with low response element copies applied to inducible mammalian gene expression. Outcome: no signal. Reason: insufficient transcription factor binding sites failed to drive inducible reporter expression
Engineering Metabolically Weaponized T Cell Therapies for Solid Tumor Translation · Georgia Tech · PhD
Tried and failed
synthetic hybrid promoters with truncated core applied to yeast gene expression. Outcome: worse than baseline. Reason: truncating the core promoter elements drastically reduced transcription compared to wild-type promoter
Enhancing secreted production from yeast species with high throughput microdroplet screening methods · UT Austin · PhD
Tried and failed
predicting expression from summative thermodynamic promoter models applied to plasmid copy number regulation. Outcome: no signal. Reason: Predicted promoter strength did not correlate with actual plasmid copy number across degenerate promoter variants
Control of Plasmid Copy Number for Synthetic Biology · Cornell · PhD
Tried and failed
tuning promoter strength to optimize reporter signal applied to transcription factor biosensors. Outcome: worse than baseline. Reason: weaker promoters severely reduced dynamic range while lowering inducer levels eliminated pathway product formation
Towards biosensor-assisted directed evolution of myo-inositol oxygenase · MIT · PhD
Lost to a baseline
pTEF promoter for HMG-CoA reductase yielded lower valencene titers than the erythritol-inducible 8UAS-pEYK promoter.
Synthetic microbial communities and multiplexed metabolic tuning by CRISPRai for enhanced bioproduction · Imperial · PhD
Lost to a baseline
Collective folding of both promoter/enhancer and gene body resulted in lower gene expression than DNA nanoparticles retaining linear duplex promoter/enhancer domains.
Novel platform for gene delivery using self-assembling DNA nanotechnology · Iowa State · PhD
Considered and rejected
Considered and rejected: Single-promoter polycistronic construct architecture for CAR and CIndR, rejected because it failed to yield robust CIndR expression (necessitating divergent dual-promoter vectors).
Constructing Hybrid Cytokine Receptors to Amplify CAR T Cell Anti-Tumor Potency · University of Washington · Doctorate
Considered and rejected
Considered and rejected: Rejected relying on native promoters en masse for heterologous expression because they are relatively weak and conditionally expressed.
Natural products isolation from Trichoderma reesei · Leibniz Universität Hannover · PhD
Considered and rejected
Considered and rejected: Rejected using intact native rtcBA promoter for in vitro transcription assays because it failed to yield detectable transcription levels; replaced with hybrid PrtcBA-nifH template.
The Rtc RNA Repair System in E. coli: underlying molecular regulation and its role in infection and antibiotic resistance. · Imperial · PhD
Tried and failed
Cross-species heterologous promoter reporter expression applied to invertebrate chordate tissue-specific transcription. Outcome: no signal. Reason: Vertebrate promoter sequences lack functional conservation or compatibility with invertebrate chordate transcriptional machinery
Investigating The Mechanisms Underlying Metamorphosis in The Chordate Ciona Robusta · Georgia Tech · PhD
Considered and rejected
Considered and rejected: Non-amplifying FLEx MyoD differentiation circuit in C3H10T1/2 cells rejected due to insufficient recombinase activation to drive phenotypic transdifferentiation without an amplification layer
Engineering chemical- and light-inducible split proteins to regulate RNA and transgene expression in mammalian cells · Boston University · PhD
Tried and failed
chimeric bacterial transcription factors for eukaryotic sensing applied to plant synthetic biosensors. Outcome: no signal. Reason: bacterial regulatory proteins failed to drive functional activation from the eukaryotic minimal promoter scaffold
Engineering plant-microbe communication for synthetic symbioses · MIT · PhD
Tried and failed
bicistronic reporter architecture for selection and screening applied to optogenetic circuit enrichment. Outcome: no signal. Reason: Bicistronic configuration compromised light-inducibility and failed to enrich target phenotypes under selection pressure
Genetic circuits for directed evolution of bioluminescent proteins · Imperial · PhD
Strong expression and protein overexpression trigger severe metabolic burden and growth defects
Driving recombinant pathways and regulators with strong constitutive promoters imposed heavy metabolic burdens on host cells. This burden resulted in growth retardation, counterselection, and the rapid accumulation of disabling mutations.
Tried and failed
combining strong constitutive promoters with strong RBS applied to transcription factor expression in bacterial circuits. Outcome: unstable. Reason: overexpression caused severe metabolic burden or toxicity, leading to selective accumulation of mutations
A synthetic biology toolbox for electrical control of gene expression · Imperial · PhD
Tried and failed
co-expressing a nonfunctional protein as negative control applied to synthetic genetic feedforward circuits. Outcome: worse than baseline. Reason: the control protein imposed an additional translational burden without providing any regulatory benefit
Modeling and controlling resource loading in bacterial genetic circuits · MIT · PhD
Tried and failed
constitutive expression of multi-gene biosynthetic pathways applied to bacterial metabolic engineering plasmids. Outcome: unstable. Reason: high metabolic burden induced genetic instability and spontaneous mutations during culturing
Investigating the potential of division of labour in synthetic bacterial communities for bioproduction · Imperial · PhD
Tried and failed
positive autoregulatory feedback with high translation initiation applied to synthetic transcriptional homeostasis circuits. Outcome: unstable. Reason: runaway expression led to toxic protein accumulation preventing successful cloning
Exploring the design principles of orthgonal transcription control systems · UT Austin · PhD
Tried and failed
plasmid-based heterologous repressor expression applied to bacterial genetic logic circuit construction. Outcome: unstable. Reason: specific repressor variants caused severe cellular toxicity and growth defects with standard ribosome binding sites
Lost to a baseline
High-copy 2μ plasmid transformants yielded lower average mNeonGreen fluorescence than low-copy CEN6/ARS4 vectors for 11 out of 12 tested Saccharomyces strains due to metabolic burden.
EXPANDING THE GENETIC TOOLKIT FOR THE CHARACTERIZATION OF DIASTATIC AND NON-TRADITIONAL YEAST STRAINS · Cornell · PhD
Considered and rejected
Considered and rejected: Native strong tac promoter rejected due to imposing cellular metabolic burden and growth defects on GFP+ cells
Defining the effects of mismatches on type I-E CRISPR immunity and phage escape · Iowa State · PhD
Considered and rejected
Considered and rejected: Rejected constitutive Anderson promoter expression for violacein pathway due to severe metabolic burden, assembly failures, and genetic instability/mutations.
Investigating the potential of division of labour in synthetic bacterial communities for bioproduction · Imperial · PhD
Considered and rejected
Considered and rejected: Rejected Syp-DTA BAC transgenic approach due to BAC copy instability/silencing and constitutive probasin promoter activation from birth causing chronic ablation and compensatory reactions.
Understanding the role of neuroendocrine cells in prostate carcinogenesis · Cornell · PhD
Considered and rejected
Considered and rejected: High-strength promoters (pTDH3/pCCW12) for driving lycopene pathway enzymes were rejected due to severe cell burden, slow growth, and plasmid/strain instability.
Characterising promoter heterogeneity for the production of novel wound healing proteins in S. cerevisiae · Imperial · PhD
Considered and rejected
Considered and rejected: Rejected plasmid-borne dCas9 delivery in P. putida due to severe metabolic burden and growth retardation
Model-driven DBTL cycle acceleration with broad-host-range bacterial CRISPRa/i circuits · University of Washington · PhD
Considered and rejected
Considered and rejected: Rejected characterising promoters in E. coli via dampened TcdR expression before transfer, because it caused cellular burden, bimodality, and diversity bottlenecks.
Tried and failed
protein overexpression from orthogonal plasmid applied to bacterial plasmid copy number control. Outcome: unstable. Reason: the orthogonal plasmid caused severe cellular fitness defects
Insights on Minimalistic Control Systems from in vivo Visualization of Plasmids · Harvard · PhD
Lost to a baseline
Insertion of an empty plasmid vector into wild-type S. oneidensis MR-1 resulted in decreased relative catalytic fluorescent turn-on compared to plasmid-free wild-type MR-1.
An investigation of inorganic nanomaterial synthesis via extracellular electron transfer by Shewanella oneidensis · UT Austin · PhD
Considered and rejected
Considered and rejected: Constitutive Ptar promoter for operon library assembly, rejected due to extreme counterselection and enrichment of broken constructs in the motile host strain.
Evolutionary optimization of bacterial motility and chemotaxis pathways Evolutionäre Optimierung bakterieller Motilitäts- und Chemotaxiswege · University of Marburg · PhD
Repetitive sequences and host propagation stress cause plasmid structural instability and mutational loss
Plasmids suffered from spontaneous deletions, multimerization, and homologous recombination during bacterial propagation and library amplification. Growth bias and cryptic promoter toxicity repeatedly degraded construct integrity across selective conditions.
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
reverting non-canonical mutations to wild-type sequences applied to bacterial plasmid replication origins. Outcome: unstable. Reason: caused severe growth defects and large spontaneous deletions in host cells
Engineered plasmids : uncovering lost histories and improving annotation · UT Austin · PhD
Tried and failed
tapering selection pressure before unselected growth applied to bacterial plasmid copy number stabilization. Reason: residual selection effects persisted and shifted plasmid copy number distributions despite antibiotic washout
Insights on Minimalistic Control Systems from in vivo Visualization of Plasmids · Harvard · PhD
Tried and failed
standard bacterial cloning at 37 C applied to repetitive Cas9 sgRNA plasmid vectors. Outcome: unstable. Reason: unintended bacterial homologous recombination degraded plasmid integrity under standard growth conditions
Dynamics and regulation of polarity proteins during epithelial polarization in primed mouse embryonic stem cells · UT Austin · PhD
Tried and failed
amplifying complex plasmid libraries in liquid culture applied to high-throughput reporter plasmid libraries. Outcome: unstable. Reason: growth bias caused a minority of clones to overtake the culture, reducing library diversity
Investigation of the Multiple Roles of Notch Signaling in Embryonic Retinal Development Using Novel High-Throughput Techniques · Harvard · PhD
Tried and failed
site-directed mutagenesis to correct plasmid mutations applied to bacterial expression plasmid construct. Reason: primers failed to produce the intact desired construct across all screened colonies
EVALUATION OF BACTERIAL BIODEGRADATION AS A MEANS TO REDUCE POLYETHYLENE POLLUTION · Johns Hopkins · Master's
Lost to a baseline
pMTL-CW22 plasmid loss efficiency (-142.48% / 53.02%) was substantially lower and more variable than pRF215 (96.23%)
Transposon mutagenesis in RT 078 Clostridioides difficile · Nottingham · PhD
Considered and rejected
Considered and rejected: Discarded plasmid clones exhibiting multimerization/dimerization to eliminate heterogeneity in fluorescence and reduce evolutionary instability.
Predicting gene expression burden to design against evolutionary failure · UT Austin · PhD
Considered and rejected
Considered and rejected: Rejected bacteria-based plasmid propagation due to instability and accumulation of cryptic-promoter-driven attenuating mutations in E. coli.
Usutu Virus: An Emerging Arbovirus Threat · Virginia Tech · Master's
Considered and rejected
Considered and rejected: Passaging plasmids containing large replication genes through dam- E. coli was rejected due to severe plasmid degradation; replaced by constructing host strains deleted for the mrr endonuclease (∆mrr).
Atypical DNA replication in Haloferax volcanii in the absence of replication origins · Nottingham · PhD
Considered and rejected
Considered and rejected: Rejected centromere-based episomal maintenance plasmids due to extreme instability and recombination of the 95% AT centromeric sequence during bacterial propagation.
Advancing Functional Genomics in P. falciparum Through Systems Biology Approaches · University of Washington · PhD
Regulatory crosstalk and contextual interference disrupt independent circuit function
Multiplexed signaling components and uninsulated adjacent cassettes suffered from transcriptional readthrough, supercoiling clashes, and signaling crosstalk. Shared cellular machinery saturation and non-specific binding prevented multi-input circuits from maintaining intended orthogonal logic.
Tried and failed
flanking with insulator sequence elements applied to tandem synthetic gene circuits. Outcome: no signal. Reason: failed to reduce or eliminate upstream transcriptional dominance mediated by supercoiling
Supercoiling as a regulator: the role of biophysical feedback in transcription and chromatin dynamics · MIT · PhD
Considered and rejected
Considered and rejected: Rejected combining two synthetic promoters sensing the same TF (or TF family) in an AND gate, as binding profiles are too similar to prevent on-target off-tumor effects.
Synthetic Genetic Circuits for Cancer Immunotherapy · Harvard · Master's
Considered and rejected
Considered and rejected: Rejected using an inducible promoter or downstream trap in circularization reporters due to disruption by transcriptional clashes and DNA supercoiling at attachment sites.
Interplay between an integrative and conjugative element and its bacterial host · MIT · PhD
Tried and failed
multiplexed quorum sensing circuits applied to orthogonal dynamic transcriptional regulation. Outcome: no signal. Reason: signal crosstalk between sender-receiver pairs caused non-responsive gene expression
Multiplexed transcriptional control strategies for biosynthesis from mixed substrates in Escherichia coli · MIT · PhD
Tried and failed
co-expressing full-length wild-type repressor and antagonist applied to synthetic genetic logic gates. Reason: wild-type oligomerization domains caused non-functional multimerization, disrupting circuit logic
Advancing Genetic Circuit Design with Prediction, Reversible Logic, and Thermal Induction · Georgia Tech · PhD
Tried and failed
multiplexed orthogonal quorum sensing systems applied to intracellular synthetic gene regulatory circuits. Reason: substantial promoter and mixed signaling crosstalk occurred when paired within a single cell
Dynamic Regulation of Metabolic Flux Using Orthogonal Quorum Sensing · MIT · PhD
Tried and failed
multi-input RNA-based logic circuits applied to mammalian cellular computation. Reason: saturation of shared endogenous RNA processing machinery by competing input substrates
Considered and rejected
Considered and rejected: Rejected direct promoter-competition ratiometric sensing because separating the integrating molecule from output makes the system sensitive to global resource fluctuations.
Design of a robust ratiometric sensor and a long-term memory genetic toggle switch · MIT · PhD
Considered and rejected
Considered and rejected: Rejected full gRNA repeat sequence occlusion by asRNA sponges in multi-node circuits to prevent crosstalk/non-orthogonality across distinct gRNAs.
Massively-parallel characterization of CRISPR nuclease binding and applications in the creation of CRISPR-based gene circuits · Cornell · PhD
Tried and failed
reversing unidirectional transcriptional terminators applied to synthetic genetic circuits. Outcome: unstable. Reason: variable transcriptional readthrough based on intrinsic terminator efficiency caused circuit inconsistency
DEVELOPING A TRANSCRIPTIONAL PROGRAMMING EDIFICE USING SYSTEMS OF ENGINEERED TRANSCRIPTION FACTORS · Georgia Tech · PhD
Considered and rejected
Considered and rejected: Rejected plasmid-based fluorescent reporter vectors for bacterial competition assays due to high-frequency plasmid exchange in vivo; required genomic recombineering.
Adherent Invasive Escherichia Coli Regulates Intestinal and Systemic Immunity in Health and Crohn's Disease · Cornell · PhD
Considered and rejected
Considered and rejected: Rejected delivering plasmid-curing elements via self-transmissible conjugative plasmids because unrestricted spread risks transferring synthetic plasmids into unintended hosts and disrupting non-target plasmids.
Advancing Tools for Quantifying and Engineering Microbial Consortia · Duke · PhD
Tried and failed
inserting ribozyme insulators to reduce contextual interference applied to synthetic multi-input logic circuits. Reason: pre-translational interactions and context effects were not mitigated by RNA cleaving insulation
Advancing Genetic Circuit Design with Prediction, Reversible Logic, and Thermal Induction · Georgia Tech · PhD
Alternative transformation and delivery approaches underperform compared to standard methods
Linearized plasmids and convective transfection methods yielded lower recombination and delivery efficiencies than conventional PCR fragments or standard chemical reagents. Membrane fragility and origin-specific variations similarly hindered transformation success in non-model hosts.
Lost to a baseline
Convective microfluidic transfection of large multicistronic CRISPR/Cas9 plasmids into iPSCs performed no better than standard Lipofectamine Stem (<1% efficiency).
Microfluidic Cell Processing for Personalized and Regenerative Medicine · Georgia Tech · PhD
Tried and failed
low-copy plasmid origins and genomic integration applied to transcription factor biosensor circuit constructs. Reason: cloning low-copy or integrated versions of the genetic circuit failed
Towards biosensor-assisted directed evolution of myo-inositol oxygenase · MIT · PhD
Lost to a baseline
Low-copy pBBR1-G159S plasmid produced lower and more variable p-AF (163 uM) compared to wild-type pBBR1 (713 uM)
Model-driven DBTL cycle acceleration with broad-host-range bacterial CRISPRa/i circuits · University of Washington · PhD
Lost to a baseline
Natural transformation frequency was 3.7-fold and 7.6-fold lower than electroporation for plasmid and chromosomal DNA, respectively.
Improved genetic tools and evolution-aware approaches for engineering honey bee gut symbionts · UT Austin · PhD
Considered and rejected
Considered and rejected: Electroporation of plasmids into P. oleovorans was rejected due to unstable performance caused by membrane fragility, and replaced with bacterial conjugation using E. coli WM3064.
Characterization of Type IV-A CRISPR-Cas systems Charakterisierung von Typ IV-A CRISPR-Cas-Systemen · University of Marburg · PhD
Continuous selection circuits fail due to host escape mutations and bypass mechanisms
Evolving proteins within selection circuits caused variants to adapt by escaping circuit repression rather than enhancing intended catalytic activity. Host strains also bypassed selection through native resistance gene activation or premature purging of target alleles.
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
spectinomycin selection for plasmid maintenance applied to Pseudomonas putida host strain. Reason: activation of native aadA resistance genes allowed growth of plasmid-free cells
Engineering the workhorse of biology : the future of protein engineering in three polypeptides · UT Austin · PhD
Considered and rejected
Considered and rejected: Rejected using helper phage/ssDNA PFunkel or chromosomal recombineering in favor of plasmid-based inverse PCR to allow strict IPTG repression and prevent premature purging of deleterious alleles during library construction.
Collateral fitness effects of mutations · Johns Hopkins · PhD
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
Left open by the authors
Problems the authors named and did not get to.
Left open
Construct a genetic circuit combining transcriptional promoter repression and TF-mediated attachment site interception to regulate recombinase activity. Blocker: Requires wet lab molecular cloning, synthetic gene circuit construction, and cellular assays
Interception of Recombinase Function: Repurposing Transcription Factors as Post-Translational Regulators of Genetic Memory · Georgia Tech · PhD
Left open
Demonstrate feedforward control coupling SpoTH directly to a toxic gene of interest such as dCas9 under the same inducible promoter. Blocker: Requires a wet biology lab and genetic engineering experimental setup to express toxic proteins and measure growth kinetics.
Modeling and controlling resource loading in bacterial genetic circuits · MIT · PhD
Left open
Integrate all synthetic cell genes onto a single genome using tuned synthetic promoters, terminators, and ribosome binding sites. Blocker: Requires a molecular biology wet lab, genetic synthesis, and cell engineering infrastructure
Left open
Construct and evaluate multi-input multi-output Transcriptional Programming logic circuits in Saccharomyces cerevisiae beyond basic BUFFER and NOT gates. Blocker: Requires a wet lab and genetic engineering of Saccharomyces cerevisiae strains.
Mapping Alternate Allosteric Communication of LacI Anti-Repressors and Their Application in Saccharomyces cerevisiae - Laying the Foundation for Designing Synthetic Transcription F · Georgia Tech · PhD
Left open
Test higher and lower vanillic acid concentrations in the bacterial circuit to measure promoter induction and Cas13a burden on cell growth. Blocker: Requires a wet biology laboratory, bacterial cell culture, and plasmids described in the thesis.
Engineering Regulatory Circuits with the Csr System and CRISPR-Cas Technology · UT Austin
Left open
Troubleshoot and investigate P1bcn and PtcdB promoter sequences for synthetic promoter library generation in Clostridium butyricum. Blocker: Requires a wet-lab biological setup and genetic engineering in Clostridium butyricum
Left open
Perform transcriptomic and proteomic assays to compare cellular resource burden between the designed genetic circuits and Cello-designed equivalents. Blocker: Requires wet lab facilities, biological samples, and sequencing/mass spectrometry equipment to generate transcriptomic and proteomic data
Genetic circuit design platforms for engineering living therapeutics · Georgia Tech · PhD
Left open
Investigate conditions where competitor sponge sites impact CRISPRi toggle switches across varying circuit affinity, promoter efficiency, and dCas12a production rates. Blocker: Requires a molecular biology wet lab, E. coli strains, and microfluidics/sequencing infrastructure to measure in vivo circuit dynamics.
ENGINEERING CRISPR-BASED TOGGLE SWITCHES IN ESCHERICHIA COLI · Cornell · PhD
Left open
Develop hierarchical Boolean circuit synthesis methods to handle designs with multiple levels of abstraction when structure is unknown. Blocker: Lack of concrete approach, target architectures, or specific synthesis strategies defined in the thesis
Learning boolean circuits from examples · Imperial · PhD
Who has worked on this
Authors of the public theses cited on this page, most-cited first.
- Prasaad Thomas Milner · Georgia Tech · PhD · 2024
Advancing Genetic Circuit Design with Prediction, Reversible Logic, and Thermal Induction - Najmia Chakri Amirina-Nave · Harvard · Master's · 2024
Drug-Responsive Regulation and Comparison of Engineered Transcription Factors Containing Different Activation Domains - Ukjin Kwon · MIT · PhD · 2023
Design of a robust ratiometric sensor and a long-term memory genetic toggle switch - Harman Mehta · Imperial · PhD · 2025
Investigating the potential of division of labour in synthetic bacterial communities for bioproduction - Valerie Ellen Kay · Georgia Tech · PhD · 2025
Mapping Alternate Allosteric Communication of LacI Anti-Repressors and Their Application in Saccharomyces cerevisiae - Laying the Foundation for Designing Synthetic Transcription Factors - Renqi Wang · Harvard · Master's · 2024
Synthetic Genetic Circuits for Cancer Immunotherapy - Rachel Lily Terry · Harvard · PhD · 2024
Insights on Minimalistic Control Systems from in vivo Visualization of Plasmids - Brian David Huang · Georgia Tech · PhD · 2024
Genetic circuit design platforms for engineering living therapeutics - Jennifer Kaczmarek Nash · MIT · PhD · 2023
Towards biosensor-assisted directed evolution of myo-inositol oxygenase - Cholpisit Kiattisewee · University of Washington · PhD · 2023
Model-driven DBTL cycle acceleration with broad-host-range bacterial CRISPRa/i circuits - Rhiannon Leyden-Preece · Imperial · PhD · 2022
Synthetic promoter library activated by alternative sigma factor enables diverse expression in Clostridium spp. and avoids limitations caused by expression during library construction - Carlos Barajas · MIT · PhD · 2022
Modeling and controlling resource loading in bacterial genetic circuits
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