addgene cidar moclo kit  (Addgene inc)

Journal: ACS Synthetic Biology

Article Title: Construction and Characterization of MoClo-Compatible Vectors for Modular Protein Expression in E. coli

doi: 10.1021/acssynbio.4c00564

Figure Lengend Snippet: Fluorescence readings of MoClo-constructed GFP constructs. (A) Normalized maximum fluorescence of E. coli BW25113 cultures carrying various GFP expression constructs grown in LB or (B) M9 minimal medium with 1% glucose per promoter, RBS, and origin of replication. Error bars represent the standard deviation of three biological replicates. (C) Range of normalized maximum fluorescence measurements of E. coli BW25113 cultures grown in LB or (D) M9 minimal medium 1% glucose per origin of replication. Black dots within bars represent individual constructs, black vertical line represents the median normalized maximum fluorescence of all constructs per origin of replication.

Article Snippet: − For E. coli , the CIDAR MoClo kit was the first modular Golden Gate DNA parts library made publicly available via Addgene (Kit #1000000059) and it has been widely used since.

Techniques: Fluorescence, Construct, Expressing, Standard Deviation

Journal: ACS Synthetic Biology

Article Title: Construction and Characterization of MoClo-Compatible Vectors for Modular Protein Expression in E. coli

doi: 10.1021/acssynbio.4c00564

Figure Lengend Snippet: Burden assessment of MoClo-compatible destination vectors with different origins of replication. E. coli BW25113 carrying various constitutive GFP expression constructs in p15A, pBR322, or pUC19 backbones were grown in LB (A, C) or M9 minimal medium with 1% glucose (B, D) and assessed for their growth rates (A, B) and maximum achieved OD 600 (C, D). Data points represent averages of biological triplicate measurements, and error bars represent their standard deviations. The visualized data is also provided in table format in Supporting Table S4 , including a distinction of the exact promoter, RBS, and backbone combination used per result.

Article Snippet: − For E. coli , the CIDAR MoClo kit was the first modular Golden Gate DNA parts library made publicly available via Addgene (Kit #1000000059) and it has been widely used since.

Techniques: Expressing, Construct

Journal: ACS Synthetic Biology

Article Title: Construction and Characterization of MoClo-Compatible Vectors for Modular Protein Expression in E. coli

doi: 10.1021/acssynbio.4c00564

Figure Lengend Snippet: Growth-coupled selection for optimal adhE * expression levels using MoClo-mediated randomized construct assembly. (A) Schematic of E. coli central metabolism depicting the catabolism of ethanol by AdhE*—the acetaldehyde intermediate is not depicted. (B) Workflow of the selection experiment starting with randomized Golden Gate Assembly of promoters, RBSs and destination vectors, yielding a diverse population of plasmids. Subsequent transformation and passaging on M9 minimal medium with 1 mM glucose + 300 mM ethanol results in selection for optimal expression of adhE *. (C) Number of sequenced clones carrying a certain adhE * expression construct before selection on ethanol medium was imposed. (D) Number of sequenced clones carrying a certain adhE * expression construct after 1, 2, or 3 passages on M9 minimal medium with 1 mM glucose + 300 mM ethanol. Three independent populations were transformed and passaged, four colonies of each population were picked at each step (pre-selection and passage 1–3) and the resulting construct composition counts were combined in the heatmaps shown in panels C and D. See Table S5 for raw counts and expression construct composition per independent population and per step. Created with Biorender.com.

Article Snippet: − For E. coli , the CIDAR MoClo kit was the first modular Golden Gate DNA parts library made publicly available via Addgene (Kit #1000000059) and it has been widely used since.

Techniques: Selection, Expressing, Construct, Transformation Assay, Passaging, Clone Assay

Journal: Nucleic acids research

Article Title: Golden Standard: a complete standard, portable, and interoperative MoClo tool for model and non-model proteobacteria.

doi: 10.1093/nar/gkad758

Figure Lengend Snippet: Figure 6. ( A ) PHB operon construction combining Golden Standard with MoClo. Le v el 0 parts for PHB production previously constructed with the MoClo toolkit were assembled in le v el 1 MoClo v ectors to obtain three TUs. Afterwards, these Tus were assembled in three le v el 2 GS pSEVA plasmids with RK2, pBBR1 or pUC origins of replication. ( B ) PHB granules present in E. coli DH10B cells harbouring plasmids pSEVA6219[gB] PHB (8% PHB), pSEVA63g19[gB] PHB (13% PHB) and pSEVA6819[gB] PHB (50% PHB). Image captured using phase contrast microscopy.

Article Snippet: We further engineered libraries f transcription units (le v el 1) e xpressing cr tE , cr tB , cr tY nd cr tZ by combina torial assembly using an equimolar ixture of selected Anderson’s promoters (J23100, J23106, 23107 and J23116) -see http://parts.igem.org/Promoters/ atalog/Anderson , obtained from the CIDAR MoClo kit Addgene Kit #1000000059).

Techniques: Construct, Microscopy

Journal: Nucleic acids research

Article Title: Golden Standard: a complete standard, portable, and interoperative MoClo tool for model and non-model proteobacteria.

doi: 10.1093/nar/gkad758

Figure Lengend Snippet: Figure 8. Schematic diagram and results of N- and C-terminal tag assemblies. ( A ) Graphical r epr esentation of individual MoClo assemblies of the OleD variants tested. ( B ) Combined plotted results (conversion of xanthohumol to 4 ′ - O - - D -glucoside of xanthohumol versus fluorescence of GFP-tag) in cell- free extracts from random clones obtained using mixed MoClo assembly with different N-terminal tags. ( C ) Graphical representation of MoClo assembly of 2xN- and 2xC protein with calculated MW masses of proteins; ( D ) SDS-PAGE analysis – on each well 1 ug of proteins was resolved. L – protein ladder; 1 – P. putida crude protein extact; 2 – eluted fraction after IMAC purification; 3 – HRV-3C digestion reaction; 4 – proteins not bound to Strep-Tactin column; 5 – proteins eluted from Strep-Tactin column. Photo gra ph of fractions 3–5 irradiated with UV light are shown.

Article Snippet: We further engineered libraries f transcription units (le v el 1) e xpressing cr tE , cr tB , cr tY nd cr tZ by combina torial assembly using an equimolar ixture of selected Anderson’s promoters (J23100, J23106, 23107 and J23116) -see http://parts.igem.org/Promoters/ atalog/Anderson , obtained from the CIDAR MoClo kit Addgene Kit #1000000059).

Techniques: Fluorescence, Clone Assay, SDS Page, Purification, Irradiation