Chapter Four · failure evidence

What Genetic Circuit & Plasmid Construction got wrong, from 69 dissertations

The records document engineering challenges encountered during genetic circuit and plasmid construction across diverse bacterial and eukaryotic systems. Common difficulties include severe metabolic burden, promoter leakiness, signal crosstalk, structural plasmid instability, and insufficient transcriptional activation. These records come from PhD theses at 20 institutions, 2021 to 2026. 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.

Basal promoter leakiness and inadequate repression cause off-state circuit failure

13 theses · 9 institutions

Uninsulated promoter contexts, shared operators, or intrinsically leaky minimal promoters produced significant baseline expression in the uninduced or repressed state. This premature activity degraded dynamic range, triggered unintended recombinase switching, and allowed cells to bypass metabolic selections.

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

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

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

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 · IRIS - SNS - prod

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

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

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

Synthetic biology tools for sensing and evolving microbes: CRISPR-diagnostics and transposon-mediated genome re-wiring · MIT

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

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

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

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

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

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

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 · OpenBU

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 · University of Nottingham Repository

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

Signal crosstalk and resource competition disrupt multi-component circuit operations

12 theses · 5 institutions

Complex genetic circuits suffered from unintended molecular interactions such as crosstalk between non-orthogonal quorum sensing systems, supercoiling interference, and promoter competition. In addition, saturation of shared cellular machinery and physical proximity of regulatory elements disrupted independent circuit logic operations.

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

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

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 · ResearchWorks

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

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

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

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

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

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

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

Scaling up Genetic Circuits in Mammalian Cells: A U1-snRNA-based Platform Enables Mammalian Cells to Compute the Bitwise Inversion of the Square Root of a Number · MIT

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

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

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

High expression burden and cellular toxicity trigger growth defects and mutations

12 theses · 9 institutions

High-strength promoters, strong ribosome binding sites, and high-copy plasmids imposed severe metabolic burden and cellular toxicity on host strains. This excessive expression caused growth defects, selection for inactivating mutations, and failure during construct cloning.

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

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

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

Genomically encoded logic gates and cell-cell communication devices for the implementation of a cryptographic hashing algorithm in living cells · MIT

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

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

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

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 · ResearchWorks

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.

Synthetic promoter library activated by alternative sigma factor enables diverse expression in Clostridium spp. and avoids limitations caused by expression during library construction · Imperial

Considered and rejected

Considered and rejected: Rejected bacterial plasmid midiprep/glycerol stock maintenance due to cryptic prokaryotic promoter toxicity causing deleterious mutations and deletions.

Development, Characterization, and Use of Molecular Tools to Study Immune-Driven Zika Virus Evolution · Virginia Tech

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

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

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 · open_UMR Marburg DSpace 10.0

Plasmid structural instability and recombination lead to construct degradation and loss

12 theses · 6 institutions

Plasmids containing repetitive sequences, AT-rich centromeric regions, or fragile replication origins experienced severe structural rearrangements, spontaneous deletions, and multimerization. Selective growth bias and inadequate antibiotic selection pressure also allowed plasmid-free cells to overtake cultures during propagation.

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

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

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

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

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

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 · JScholarship

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 · University of Nottingham Repository

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

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 · University of Nottingham Repository

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 · ResearchWorks

Considered and rejected

Considered and rejected: Decided against serial D2O adaptation across multiple passages in favor of direct transfers to avoid plasmid instability in recA+/endA+ hosts.

Revisiting Allostery In Lac Repressor · Penn

Weak promoter activity and core incompatibility prevent adequate gene expression

11 theses · 8 institutions

Engineered promoters frequently failed to produce sufficient expression levels due to truncated core elements, lack of essential motifs like Y-patches, or low operator copy numbers. Similarly, heterologous or bacterial transcription factors were unable to function effectively across divergent host transcriptional machineries.

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

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

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

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

Tried and failed

pairing distal cis-regulatory modules with canonical TATA-box promoters applied to cell-type-specific synthetic promoter design. Outcome: no signal. Reason: canonical TATA core promoters lacked essential Y-patch elements required for distal module activation

A transcription factor ensemble orchestrates bundle sheath expression in rice. · Cambridge

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 Repository

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

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

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 · OpenBU

Considered and rejected

Considered and rejected: Decided against using the CMV promoter in rAAV constructs due to its susceptibility to epigenetic silencing via DNA methylation and histone deacetylation in specific tissues over time.

Molecular and Ocular Characterization of Novel Fibulin-3 Variants Involved in Retinal Degeneration · DSpace at UTSWMED

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

Suboptimal delivery and physical recombination limit plasmid introduction efficiency

8 theses · 7 institutions

Methods for introducing or integrating plasmid DNA encountered low delivery efficiencies, cellular membrane fragility, and suboptimal recombination frequencies compared to linear cassettes. In other instances, unintended horizontal plasmid transfer or poor transformant performance limited experimental utility.

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

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

Lost to a baseline

Linearized plasmid DNA achieved ~100-fold lower recombination frequency compared to PCR-amplified linear recombination cassettes.

Expanding the bio-capabilities of Lactococcus lactis through efficient genome modification · Iowa State

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 · ResearchWorks

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

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 · open_UMR Marburg DSpace 10.0

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

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 · DukeSpace

Selection evasion and screening artifacts undermine directed evolution circuits

3 theses · 2 institutions

Genetic selection and continuous directed evolution circuits failed when variants evolved to escape the circuit mechanism rather than improving target catalytic activity. In addition, bicistronic reporter configurations compromised induction responsiveness under selection pressure.

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

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

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

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

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

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

Towards an artificial cell: Development of a system which self-regenerates the protein components of the PURE system in microfluidic reactors · EPFL

Left open

Test whether the bundle sheath cis-regulatory module functions via enhanceosome or billboard motif grammar using reporter assays. Blocker: Requires wet lab molecular biology experiments, plant transformations, and reporter assays to test synthetic promoter variants.

A transcription factor ensemble orchestrates bundle sheath expression in rice. · Cambridge

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

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

Synthetic promoter library activated by alternative sigma factor enables diverse expression in Clostridium spp. and avoids limitations caused by expression during library construct · Imperial

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

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

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

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