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Showing 1 - 50 of 178385 result(s)
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Public
Glucoamylase-aMFΔ
Glucoamylase_u45_aMF_u916_ Version 1 (Component)
Glucoamylase followed by aMFΔ
Public
Inulinase-aMFΔ
Inulinase_u45_aMF_u916_ Version 1 (Component)
Inulinase followed by aMFΔ
Public
Invertase-aMFΔ
Invertase_u45_aMF_u916_ Version 1 (Component)
Invertase followed by aMFΔ
Public
Killer-aMFΔ
Killer_u45_aMF_u916_ Version 1 (Component)
Killer followed by aMFΔ
Public
PARS
PARS Version 1 (Component)
Pichia autonomously replicating sequence
Public
RFP
RFP Version 1 (Component)
Red fluorescent protein, BsaI removed
Public
RFP_Sec
RFP_Sec Version 1 (Component)
Red fluorescent protein, BsaI removed
Public
SA-aMFΔ
SA_u45_aMF_u916_ Version 1 (Component)
Serum albumin followed by aMFΔ
Public
aAmylase-aMFΔ
aAmylase_u45_aMF_u916_ Version 1 (Component)
α-Amylase followed by aMFΔ
Public
aMF
aMF Version 1 (Component)
α-mating factor
Public
aMF_no_EAEA
aMF_no_EAEA Version 1 (Component)
α-mating factor no EAEA
Public
aMFΔ
aMF_u916_ Version 1 (Component)
α-mating factor, pre-sequence shortened
Public
aMFΔ_no_Kex
aMF_u916__no_Kex Version 1 (Component)
α-mating factor no Kex recognition site
Public
attB
attB Version 1 (Component)
BxbI recognition site, BsaI site removed
Public
pAOX1
pAOX1 Version 1 (Component)
Alcohol oxidase 1
Public
pENO1
pENO1 Version 1 (Component)
Enolase 1
Public
pGAP
pGAP Version 1 (Component)
Glyceraldehyde-3-phosphate dehydrogenase
Public
pTPI1
pTPI1 Version 1 (Component)
Triose phosphate isomerase 1
Public
tAOX1
tAOX1 Version 1 (Component)
Alcohol oxidase 1
Public
yEGFP
yEGFP Version 1 (Component)
Green fluorescent protein
Public
yEGFP_Sec
yEGFP_Sec Version 1 (Component)
Green fluorescent protein
Public
Pichia MoClo Toolkit (Lu Lab)
Pichia_MoClo_Toolkit_Lu_Lab_collection Version 1 (Collection)
Pichia MoClo Toolkit (Lu Lab)
Public
Engineered Production of Short-Chain Acyl-Coenzyme A Esters in Saccharomyces cerevisiae
sb7b00466 Version 1 (Collection)
Short-chain acyl-coenzyme A esters serve as intermediate compounds in fatty acid biosynthesis, and the production of polyketides, biopolymers and other value-added chemicals. S. cerevisiae is a model organism that has been utilized for the biosynthesis of such biologically and economically valuable compounds. However, its limited repertoire of short-chain acyl-CoAs effectively prevents its application as a production host for a plethora of natural products. Therefore, we introduced biosynthetic metabolic pathways to five different acyl-CoA esters into S. cerevisiae. Our engineered strains provide the following acyl-CoAs: propionyl-CoA, methylmalonyl-CoA, n-butyryl-CoA, isovaleryl-CoA and n-hexanoyl-CoA. We established a yeast-specific metabolite extraction protocol to determine the intracellular acyl-CoA concentrations in the engineered strains. Propionyl-CoA was produced at 4–9 μM; methylmalonyl-CoA at 0.5 μM; and isovaleryl-CoA, n-butyryl-CoA, and n-hexanoyl-CoA at 6 μM each. The acyl-CoAs produced in this study are common building blocks of secondary metabolites and will enable the engineered production of a variety of natural products in S. cerevisiae. By providing this toolbox of acyl-CoA producing strains, we have laid the foundation to explore S. cerevisiae as a heterologous production host for novel secondary metabolites.
Public
A Dynamic Model of Resource Allocation in Response to the Presence of a Synthetic Construct
sb8b00015 Version 1 (Collection)
Introducing synthetic constructs into bacteria often carries a burden that leads to reduced fitness and selective pressure for organisms to mutate their constructs and hence to a reduced functional lifetime. Understanding burden requires suitable methods for accurate measurement and quantification. We develop a dynamic growth model from physiologically relevant first-principles that allows parameters relevant to burden to be extracted from standard growth curves. We test several possibilities for the response of a bacterium to a new environment in terms of resource allocation. We find that burden manifests in the time taken to respond to new conditions as well as the rate of growth in exponential phase. Furthermore, we see that the presence of a synthetic construct hastens the reduction of ribosomes when approaching stationary phase, altering memory effects from previous periods of growth.
Public
An Automated Induction Microfluidics System for Synthetic Biology
sb8b00025 Version 1 (Collection)
The expression of a recombinant gene in a host organism through induction can be an extensively manual and labor-intensive procedure. Several methods have been developed to simplify the protocol, but none has fully replaced the traditional IPTG-based induction. To simplify this process, we describe the development of an autoinduction platform based on digital microfluidics. This system consists of a 600 nm LED and a light sensor to enable the real-time monitoring of  the optical density (OD) samples coordinated with the semicontinuous mixing of a bacterial culture. A hand-held device was designed as a microbioreactor to culture cells and to measure the OD of the bacterial culture. In addition, it serves as a platform for the analysis of regulated protein expression in E. coli without the requirement of standardized well-plates or pipetting-based platforms. Here, we report for the first time, a system that offers great convenience without the user to physically monitor the culture or to manually add inducer at specific times. We characterized our system by looking at several parameters (electrode designs, gap height, and growth rates) required for an autoinducible system. As a first step, we carried out an automated induction optimization assay using a RFP reporter gene to identify conditions suitable for our system. Next, we used our system to identify active thermophilic β-glucosidase enzymes that may be suitable candidates for biomass hydrolysis. Overall, we believe that this platform may be useful for synthetic biology applications that require regulating and analyzing expression of heterologous genes for strain optimization.
Public
Engineering Translational Resource Allocation Controllers: Mechanistic Models, Design Guidelines, and Potential Biological Implementations
sb8b00029 Version 1 (Collection)
The use of orthogonal ribosomes in combination with dynamic resource allocation controllers is a promising approach for relieving the negative effects of cellular resource limitations on the modularity of synthetic gene circuits. Here, we develop a detailed mechanistic model of gene expression and resource allocation, which when simplified to a tractable level of complexity, allows the rational design of translational resource allocation controllers. Analysis of this model reveals a fundamental design trade-off: that reducing coupling acts to decrease gene expression. Through a sensitivity analysis of the experimentally tunable controller parameters, we identify how each controller design parameter affects the overall closed-loop behavior of the system, leading to a detailed set of design guidelines for optimally managing this trade-off. On the basis of our designs, we evaluated a number of alternative potential experimental implementations of the proposed system using commonly available biological components. Finally, we show that the controller is capable of dynamically allocating ribosomes as needed to restore modularity in a number of more complex synthetic circuits, such as the repressilator, and activation cascades composed of multiple interacting modules.
Public
A Biosensor Strategy for E. coli Based on Ligand-Dependent Stabilization
sb8b00052 Version 1 (Collection)
The engineering of microorganisms to monitor environmental chemicals or to produce desirable bioproducts is often reliant on the availability of a suitable biosensor. However, the conversion of a ligand-binding protein into a biosensor has been difficult. Here, we report a general strategy for generating biosensors in Escherichia coli that act by ligand-dependent stabilization of a transcriptional activator and mediate ligand concentration-dependent expression of a reporter gene. We constructed such a biosensor by using the lac repressor, LacI, as the ligand-binding domain and fusing it to the Zif268 DNA-binding domain and RNA polymerase omega subunit transcription-activating domain. Using error-prone PCR mutagenesis of lacI and selection, we identified a biosensor with multiple mutations, only one of which was essential for biosensor behavior. By tuning parameters of the assay, we obtained a response dependent on the ligand isopropyl β-d-1-thiogalactopyranoside (IPTG) of up to a 7-fold increase in the growth rate of E. coli. The single destabilizing mutation combined with a lacI mutation that expands ligand specificity to d-fucose generated a biosensor with improved response both to d-fucose and to IPTG. However, a mutation equivalent to the one that destabilized LacI in either of two structurally similar periplasmic binding proteins did not confer ligand-dependent stabilization. Finally, we demonstrated the generality of this method by using mutagenesis and selection to engineer another ligand-binding domain, MphR, to function as a biosensor. This strategy may allow many natural proteins that recognize and bind to ligands to be converted into biosensors.
Public
Targeted Repression of Essential Genes To Arrest Growth and Increase Carbon Partitioning and Biofuel Titers in Cyanobacteria
sb8b00056 Version 1 (Collection)
Photoautotrophic production of fuels and chemicals by cyanobacteria typically gives lower volumetric productivities and titers than heterotrophic production. Cyanobacteria cultures become light limited above an optimal cell density, so that this substrate is not supplied to all cells sufficiently. Here, we investigate genetic strategies for a two-phase cultivation, where biofuel-producing Synechocystis cultures are limited to an optimal cell density through inducible CRISPR interference (CRISPRi) repression of cell growth. Fixed CO2 is diverted to ethanol or n-butanol. Among the most successful strategies was partial repression of citrate synthase gltA. Strong repression (>90%) of gltA at low culture densities increased carbon partitioning to n-butanol 5-fold relative to a nonrepression strain, but sacrificed volumetric productivity due to severe growth restriction. CO2 fixation continued for at least 3 days after growth was arrested. By targeting sgRNAs to different regions of the gltA gene, we could modulate GltA expression and carbon partitioning between growth and product to increase both specific and volumetric productivity. These growth arrest strategies can be useful for improving performance of other photoautotrophic processes.
Public
Glucoamylase-aMFΔ Sequence
Glucoamylase_u45_aMF_u916__sequence Version 1 (Sequence)

Public
Inulinase-aMFΔ Sequence
Inulinase_u45_aMF_u916__sequence Version 1 (Sequence)

Public
Invertase-aMFΔ Sequence
Invertase_u45_aMF_u916__sequence Version 1 (Sequence)

Public
Killer-aMFΔ Sequence
Killer_u45_aMF_u916__sequence Version 1 (Sequence)

Public
PARS Sequence
PARS_sequence Version 1 (Sequence)

Public
RFP_Sec Sequence
RFP_Sec_sequence Version 1 (Sequence)

Public
RFP Sequence
RFP_sequence Version 1 (Sequence)

Public
SA-aMFΔ Sequence
SA_u45_aMF_u916__sequence Version 1 (Sequence)

Public
aAmylase-aMFΔ Sequence
aAmylase_u45_aMF_u916__sequence Version 1 (Sequence)

Public
aMF_no_EAEA Sequence
aMF_no_EAEA_sequence Version 1 (Sequence)

Public
aMF Sequence
aMF_sequence Version 1 (Sequence)

Public
aMFΔ_no_Kex Sequence
aMF_u916__no_Kex_sequence Version 1 (Sequence)

Public
aMFΔ Sequence
aMF_u916__sequence Version 1 (Sequence)

Public
attB Sequence
attB_sequence Version 1 (Sequence)

Public
pAOX1 Sequence
pAOX1_sequence Version 1 (Sequence)

Public
pENO1 Sequence
pENO1_sequence Version 1 (Sequence)

Public
pGAP Sequence
pGAP_sequence Version 1 (Sequence)

Public
pTPI1 Sequence
pTPI1_sequence Version 1 (Sequence)

Public
tAOX1 Sequence
tAOX1_sequence Version 1 (Sequence)

Public
yEGFP_Sec Sequence
yEGFP_Sec_sequence Version 1 (Sequence)

Public
yEGFP Sequence
yEGFP_sequence Version 1 (Sequence)

Public
pET_RFP_seq
ComponentDefinition_sb8b00025_9_comp Version 1 (Component)

Showing 1 - 50 of 178385 result(s)
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